Antibody-sting agonist conjugates and their use in immunotherapy

ABSTRACT

The present disclosure relates to, among other things, antibody-drug conjugates comprising a STING agonist cyclic di-nucleotide conjugated to an antibody, preparation methods therefor, and uses therefor.

1. SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jul. 17, 2020, isnamed 400160_005US_SL_ST25.txt and is 44,502 bytes in size.

2. FIELD

This disclosure pertains to, among other things, the use of STINGagonists in antibody-drug conjugates (ADCs) for immunotherapy;compositions including the ADCs, methods of making the ADCs, and methodsof using the ADCs to treat cancers.

3. BACKGROUND

Cytosolic DNA sensing pathway plays pivotal roles in initiation andmaintenance of immune responses against malignancies, in addition to itsprimary function in host defense against invasion of DNA-containingmicrobes. Cytosolic DNA from damaged tumor cells triggers activation ofan enzyme named cyclic AMP-GMP synthase (cGAS), which synthesizes2′3′-cyclic AMP-GMP (cGAMP). As an endogenous ligand, cGAMP binds to andactivates the ER adaptor protein Stimulator of Interferon Genes (STING),and leads to induction of interferons and inflammatory cytokines,recruitment and maturation of antigen presenting cells, and ultimatelyanti-tumor immunity carried out by T cells and natural killer (NK)cells. A number of STING agonists, which resemble cGAMP but possessimproved therapeutic properties, are under development for cancerimmunotherapy. Although these drugs are promising for treatment of arange of solid tumors, they have obvious limitations. These compoundsrely on intratumoral administration, due to their potential to inducesystemic cytokine response if injected otherwise. Even intratumoralinjection could still induce unwanted cytokine response because therapid leakage to peripheral tissues. The short exposure of thesecompounds to immune cells in tumor environment also reduces theireffect. Therefore, novel therapeutics that can specifically deliver aSTING agonist to the tumor environment with prolonged retention time arein urgent need.

4. SUMMARY

The disclosure provides particular cyclic di-nucleotides (CDNs) that canbe conjugated to antibodies or antigen-binding fragments targetingspecific antigens in the microenvironment of diseased cells or tissue.For instance, the antibodies or antigen-binding fragments thereof cantarget cancer-related antigens present on cells in the tumormicroenvironment, such as tumor cells or immune cells. The CDNs of thedisclosure are capable of agonizing STING, hence stimulating the immunesystem. When the CDNs of the disclosure are conjugated to antibodiestargeting antigens in diseased cells or tissue, they provide sufficientexposure of the CDNs in the microenvironment of diseased cells or tissuewhile reducing concomitant side effects associated with extensive CDNleakage into peripheral tissues.

In one aspect, the disclosure provides antibody-drug conjugates (ADCs)having the structure of Formula I:Ab-[-L-(D)_(m)]_(n)  (Formula I)wherein:

“D” represents a CDN (e.g., a CDN as described herein, such as those ofFormula II);

“Ab” represents an antibody or binding fragment thereof which binds atarget antigen;

“L” represents, independently for each occurrence, a linker linking oneor more occurrences of D to Ab;

“m” represents the number of occurrences of D linked to a given linker;and

“n” represents the number of linkers linked to Ab.

In certain embodiments, the disclosure provides ADCs having thestructure of Formula Ta:Ab-[-L-D]_(n)  (Formula Ia)wherein:

“D” represents a CDN (e.g., a CDN as described herein, such as those ofFormula II);

“Ab” represents an antibody or binding fragment thereof which binds atarget antigen;

“L” represents, independently for each occurrence, a linker linking D toAb; and

“n” represents the number of occurrences of D linked to Ab via thelinker (L).

In another aspect, the disclosure provides specific CDNs (D) that can beadministered by themselves or as part of the ADC of Formula I. TheseCDNs can have the structure of Formula II:

wherein

R¹ is C₁₋₆alkyl, such as C₂₋₆alkyl or C₂₋₃alkyl, substituted with ahydroxyl, thiol, amino, C₁₋₆alkylamino, or a -PEG-OH group;

R³ and R⁴ are independently hydrogen, halogen, C₁₋₆alkyl, C₂₋₆alkenyl,or C₂₋₆alkynyl, wherein C₁₋₆alkyl, C₂₋₆alkenyl, and C₂₋₆alkynyl are,independently, optionally substituted with one or more groups selectedfrom halogen, thiol, hydroxyl, carboxyl, C₁₋₆alkoxy, C₁₋₆hydroxyalkoxy,—OC(O)C₁₋₆alkyl, —N(H)C(O)C₁₋₆alkyl, —N(C₁₋₃alkyl)C(O)C₁₋₆alkyl, amino,C₁₋₆alkylamino, di(C₁₋₆alkyl)amino, oxo, and azido;

R², R⁵, and R⁶ are independently hydrogen, halogen, hydroxyl, azido,amino, C₁₋₆alkylamino, di(C₁₋₆alkyl)amino, C₁₋₆alkyl, C₁₋₆alkoxy,C₂₋₆alkenyl, C₃₋₆alkenyl-O—, C₂₋₆alkynyl, or C₃₋₆alkynyl-O—, whereinC₁₋₆alkyl, C₁₋₆alkoxy, C₂₋₆alkenyl, C₃₋₆alkenyl-O—, C₂₋₆alkynyl, andC₃₋₆alkynyl-O—, are, independently, optionally substituted with one ormore groups selected from halogen, thiol, hydroxyl, carboxyl,C₁₋₆alkoxy, C₁₋₆hydroxyalkoxy, —OC(O)C₁₋₆alkyl, —N(H)C(O)C₁₋₆alkyl,—N(C₁₋₃alkyl)C(O)C₁₋₆alkyl, amino, C₁₋₆alkylamino, di(C₁₋₆alkyl)amino,oxo, and azido; or R⁶ and R⁵ together are ═CH₂; or R⁶ and R⁴ togetherform a bridge across the ring containing V² selected from ethylene,—O—CH₂—, and —NH—CH₂—;

V¹ and V² are independently O, S, or CH₂;

BG¹, starting from the carbon in the ring containing V¹, and BG²,starting from the carbon in the ring containing V², are independently—O—P(O)R^(p)—O—, —O—P(S)R^(p)—O—, —O—P(O)R^(p)—S—, —O—P(S)R^(p)—S—,—S—P(O)R^(p)—O—, —S—P(S)R^(p)—O—, —S—P(O)R^(p)—S—, —S—P(S)R^(p)—S—, or—NH—SO₂—NH—; wherein

-   -   R^(p) is, independently for each occurrence, hydroxyl, thiol,        C₁₋₆alkyl, C₁₋₆alkoxy, C₃₋₆alkenyl-O—, C₃₋₆alkynyl-O—, -PEG-OH,        borano (—BH₃ ⁻), or —NR′R″, wherein C₁₋₆alkyl, C₁₋₆alkoxy,        C₃₋₆alkenyl-O—, and C₃₋₆alkynyl-O—, are, independently,        optionally substituted with one or more groups selected from        halogen, thiol, hydroxyl, carboxyl, C₁₋₆alkoxy,        C₁₋₆hydroxyalkoxy, —OC(O)C₁₋₆alkyl, —N(H)C(O)C₁₋₆alkyl,        —N(C₁₋₃alkyl)C(O)C₁₋₆alkyl, amino, C₁₋₆alkylamino,        di(C₁₋₆alkyl)amino, oxo, and azido; and    -   R′ and R″ are independently hydrogen or C₁₋₆alkyl optionally        substituted with one or more groups selected from halogen,        thiol, hydroxyl, carboxyl, C₁₋₆alkoxy, C₁₋₆hydroxyalkoxy,        —OC(O)C₁₋₆alkyl, —N(H)C(O)C₁₋₆alkyl, —N(C₁₋₃alkyl)C(O)C₁₋₆alkyl,        amino, C₁₋₆alkylamino, di(C₁₋₆alkyl)amino, oxo, and azido; or R′        and R″ together on the same nitrogen form a C₃₋₅heterocyclic        ring;

R^(a1), R^(b1), R^(a2), and R^(b2) are independently hydrogen orC₁₋₃alkyl; and

B¹ and B² are independently selected from:

-   -   wherein    -   V³ is O or S, particularly O;    -   Z¹, Z², Z³, Z⁴, Z⁵, and Z⁶ are, independently for each        occurrence, CR^(z) or N;    -   Z^(a) is O (except when Z⁵ is N) or NR′; wherein        -   R^(z) is, independently for each occurrence, hydrogen,            halogen, azido, amino, C₁₋₆alkylamino, di(C₁₋₆alkyl)amino,            C₁₋₆alkyl, C₁₋₆alkoxy, C₂₋₆alkenyl, C₃₋₆alkenyl-O—,            C₂₋₆alkynyl, C₃₋₆alkynyl-O—, —NO₂, —CN, —C(O)C₁₋₆alkyl,            —CO₂H, —CO₂C₁₋₆alkyl, —S(O)C₁₋₆alkyl, —S(O)₂C₁₋₆alkyl,            —C(O)NR′, —C(O)NR′R″, —SO₂NR′R″, —OC(O)C₁₋₆alkyl,            —NR′C(O)C₁₋₆alkyl, —N(R′)C(O)NR′R″, —N(R′)SO₂NR′R″,            —N(R)SO₂C₁₋₆alkyl, or —OC(O)NR′R″, wherein        -   C₁₋₆alkyl, C₁₋₆alkoxy, C₂₋₆alkenyl, C₃₋₆alkenyl-O—,            C₂₋₆alkynyl, and C₃₋₆alkynyl-O—, are, independently for each            occurrence, optionally substituted with one or more groups            selected from halogen, thiol, hydroxyl, carboxyl,            C₁₋₆alkoxy, C₁₋₆hydroxyalkoxy, —OC(O)C₁₋₆alkyl,            —N(H)C(O)C₁₋₆alkyl, —N(C₁₋₃alkyl)C(O)C₁₋₆alkyl, amino,            C₁₋₆alkylamino, di(C₁₋₆alkyl)amino, oxo, and azido; and        -   R′ and R″ are, independently for each occurrence, hydrogen            or C₁₋₆alkyl optionally substituted with one or more groups            selected from halogen, thiol, hydroxyl, carboxyl,            C₁₋₆alkoxy, C₁₋₆hydroxyalkoxy, —OC(O)C₁₋₆alkyl,            —N(H)C(O)C₁₋₆alkyl, —N(C₁₋₃alkyl)C(O)C₁₋₆alkyl, amino,            C₁₋₆alkylamino, di(C₁₋₆alkyl)amino, oxo, and azido; or R′            and R″ on the same nitrogen together form a C₃₋₅heterocyclic            ring;            or a pharmaceutically acceptable salt thereof.

The disclosure provides methods of making ADCs of formula I byconjugating a CDN of Formula II to an antibody via a linker. The CDN ofFormula II can be conjugated to the antibody via a cleavable ornon-cleavable linker. In particular embodiments, the CDN is releasedinto a tumor cell, a cancer-related immune cell, or into the tumormicroenvironment upon cleavage of the linker.

In the ADCs of Formula I, wherein the CDN (D) is of Formula II, the CDNmay be covalently bound to linker (L) at the hydroxyl, thiol, amino,C₁₋₆alkylamino, or -PEG-OH group at the R¹ position of the CDN ofFormula II.

In one embodiment of the disclosure, the CDN of Formula II has theFormula IIe:

wherein R¹, R⁵, and R⁶; R^(p); and B¹ and B² are as defined above forFormula II;or a pharmaceutically acceptable salt thereof.

In one embodiment of the disclosure, the CDN of Formula II has theFormula IIk:

wherein

W, X, Y, and Z are independently CH or N; and

R^(p), independently for each occurrence, is as defined above forFormula II;

or a pharmaceutically acceptable salt thereof.

In some embodiments, the disclosure provides a composition comprising aCDN and a base, wherein the CDN is a compound of Formula II, such asFormula IIe or Ilk above, or Formula IIn or IIo below. In certain ofsuch embodiments, the composition consists of the CDN and the base. Insome embodiments, the base is an amine base, such as pyridine. In someof these embodiments, the composition is anhydrous.

In certain embodiments, the disclosure provides a composition comprisinga CDN and a linker or a coupling agent, or both a linker and a couplingagent, wherein the CDN is a compound of Formula II, such as Formula IIeor Ilk above or Formula IIn or IIo below, and wherein the coupling agentfacilitates coupling of the CDN to the linker, for example, bygenerating an activated ester on the linker. In some embodiments, thecomposition further comprises an aprotic polar solvent. In certainembodiments, the composition is anhydrous.

In some embodiments, the disclosure provides a compound that is a cyclicdinucleotide (CDN) coupled to a linker (L) of the formula L-CDN. Incertain embodiments, the CDN is coupled to a linker L via a thioether,an amide, an ester, a carbamate, a carbonate, a urea, a disulfide, or anether group, particularly an amide, a carbamate, or a disulfide group.In certain embodiments, the CDN is of Formula II, such as Formula IIe orIlk above, or Formula IIm, IIn or IIo below, and the CDN is coupled to Lat the thiol, amino, or C₁₋₆alkylamino group of R¹ of Formula II, and Lincludes a site capable of coupling to a complementary site on anantibody.

In one embodiment, the ADC of Formula I has the structure of FormulaIII:

wherein variables W, X, Y, Z, R^(p), and n are defined as above forFormulas I and II.

In one embodiment, the ADC of Formula I has the structure of Formula IV:

wherein variables W, X, Y, Z, R^(p), and n are defined as above FormulasI and II.

In one embodiment, the ADC of Formula I or III is derived from a CDN (D)having the following structure (CDN-A):

or a pharmaceutically acceptable salt thereof. It will be understoodthat the phrase “derived from” indicates that the amino (—NH₂)functionality at the R¹ position of the CDN is covalently bound to acorresponding position in the linker. For instance, in some embodiments,the amino group is covalently bonded to a carbonyl moiety of the linker,hence forming an amide or carbamate bond.

In another embodiment, the ADC of Formula I or IV is derived from a CDN(D) having the following structure (CDN-B):

or a pharmaceutically acceptable salt thereof. It will be understoodthat the phrase “derived from” indicates that the thiol (—SH)functionality at the R¹ position of the CDN is covalently bound to acorresponding position in the linker. For instance, in some embodiments,the thiol group of the CDN is covalently bonded to a thiol group of thelinker, hence forming a disulfide bond.

In another embodiment, the ADC of Formula I or III is derived from a CDN(D) having the following structure:

or a pharmaceutically acceptable salt thereof.

In another embodiment, the ADC of Formula I or III is derived from a CDN(D) having the following structure:

or a pharmaceutically acceptable salt thereof.

In another embodiment, the ADC of Formula I or III is derived from a CDN(D) having the following structure:

or a pharmaceutically acceptable salt thereof.

In certain embodiments, the antibody or antigen-binding fragment of theADC of Formula I, III, or IV targets a specific antigen that isexpressed on tumor cells or immune cells in the tumor microenvironment.In particular embodiments, the antibody or antigen-binding fragment ofthe ADC of Formula I, III, or IV targets the receptor PD-L1. In otherparticular embodiments, the antibody or antigen-binding fragment thereofspecifically binds to a cancer related tumor antigen which is a GrowthFactor Receptor (GFR). In certain embodiments, the cancer related tumorantigen is an EGFR/ErbB/HER family GFR.

In one aspect, the disclosure provides methods of inducing an immuneresponse in a subject (e.g., a human patient) by administering atherapeutically effective amount of an ADC of Formula I, III, or IV. Forinstance, an ADC of Formula I, III, or IV can be employed for inducinginterferon-β (IFNβ) in a human subject.

The ADC of Formula I, III, or IV can be used in combination with one ormore additional therapeutic agents. The additional therapeutic agent(s)can be administered prior to, concurrently or following administrationof the additional therapeutic agent(s). In a particular embodiment, theADC of Formula I, III, or IV can be used in combination with an immunecheckpoint inhibitor. For instance, the ADC of Formula I, III, or IV canbe administered with an inhibitor of PD-1, PD-L1, or CTLA-4, or acombination thereof.

In another embodiment, the ADC of Formula I, III, or IV can beadministered with a free CDN that is not conjugated to the antibody orantigen-binding fragment of Formula I. In such cases, the free CDN maybe the same or different than the CDN that is conjugated to the antibodyof the ADC of Formula I, III, or IV.

5. BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1D show the structural characterization and activity of ADC-I.FIG. 1A shows the chemical structure of ADC-I. FIGS. 1B and 1C show thepotency of IFN stimulatory activity in a luciferase reporter assay usingmouse RAW-Lucia ISG cells, and human THP1-Lucia ISG cells, respectively.FIG. 1D shows tumor progression in B16-F10 tumor-bearing C57BL6 miceunder indicated treatments. The comparator anti-PDL1 antibody is Ab-A1.

FIGS. 2A-2C show the structural characterization and activity of ADC-II.FIG. 2A shows the chemical structure of ADC-II. FIGS. 2B and 2C show thepotency of IFN stimulatory activity in a luciferase reporter assay usingmouse RAW-Lucia ISG cells, and human THP1-Lucia ISG cells, respectively.

FIGS. 3A-3B show the structural characterization and activity ofADC-III. FIG. 3A shows the chemical structure of ADC-III. FIG. 3B showsthe potency of IFN stimulatory activity in a luciferase reporter assayusing mouse RAW-Lucia ISG cells. The comparator anti-EGFR antibody isAb-B1.

FIGS. 4A-4F show the structural characterization and activity of ADC-IV.FIG. 4A shows the chemical structure of ADC-IV. FIG. 4B shows thepotency of IFN stimulatory activity in a luciferase reporter assay usingmouse RAW-Lucia ISG cells (fold change versus PBS-stimulated cells).FIGS. 4C and 4D show tumor progression and survival, respectively, inEGFR-expressing B16F10 tumor bearing C57BL6 mice under indicatedtreatments. FIGS. 4E and 4F show tumor progression and survival,respectively, in EGFR-expressing B16F10 tumor bearing C57BL6 mice underindicated treatments. The comparator anti-EGFR antibody is Ab-B1, andthe comparator anti-PDL1 antibody is Ab-A3.

FIGS. 5A-5B show the structural characterization and activity of ADC-V.FIG. 5A shows the chemical structure of ADC-V. FIG. 5B shows the potencyof IFN stimulatory activity in a luciferase reporter assay using mouseRAW-Lucia ISG cells. The comparator anti-PDL1 antibody is Ab-A3.

FIGS. 6A-6F show the structural characterization and activity of ADC-VI.FIG. 6A shows the chemical structure of ADC-VI. FIG. 6B shows thepotency of IFN stimulatory activity in a luciferase reporter assay usingmouse RAW-Lucia ISG cells. FIGS. 6C and 6D show tumor progression andsurvival, respectively, in EGFR-expressing B16F10 tumor bearing C57BL6mice under indicated treatments. The comparator anti-PDL1 antibody isAb-A2. FIGS. 6E and 6F show tumor progression and survival,respectively, in EGFR-expressing B16F10 tumor bearing C57BL6 mice underindicated treatments.

FIGS. 7A-7F show the structural characterization and activity ofADC-VII. FIG. 7A shows the chemical structure of ADC-VII. FIG. 7B showsthe potency of IFN stimulatory activity in a luciferase reporter assayusing mouse RAW-Lucia ISG cells (fold change versus PBS-stimulatedcells). FIGS. 7C and 7D show tumor progression and survival,respectively, in EGFR-expressing B16F10 tumor bearing C57BL6 mice underindicated treatments. I.p. (intraperitoneal) injections occurred on days7, 11, and 15. FIGS. 7E and 7F show tumor progression and survival,respectively, in EGFR-expressing B16F10 tumor bearing C57BL6 mice underindicated treatments. I.p. injections occurred on days 7, 11, and 15.The comparator anti-EGFR antibody is Ab-B2, and the comparator anti-PDL1antibody is Ab-A3.

FIGS. 8A-8D show the structural characterization and activity ofADC-VIII. FIG. 8A shows the chemical structure of ADC-VIII. FIG. 8Bshows the potency of IFN stimulatory activity in a luciferase reporterassay using mouse RAW-Lucia ISG cells. FIGS. 8C and 8D show tumorprogression and survival, respectively, in EGFR-expressing B16F10 tumorbearing C57BL6 mice under indicated treatments. I.p. injections occurredon days 7, 11, and 15. The comparator anti-PDL1 antibody is Ab-A3.

FIGS. 9A-9D show the structural characterization and activity of ADC-IX.FIG. 9A shows the chemical structure of ADC-IX. FIG. 9B shows thepotency of IFN stimulatory activity in a luciferase reporter assay usingmouse RAW-Lucia ISG cells. FIGS. 9C and 9D show tumor progression andsurvival, respectively, in EGFR-expressing B16F10 tumor bearing C57BL6mice under indicated treatments. I.p. injections occurred on days 6, 9,and 13. The comparator anti-PDL1 antibody is Ab-A3.

FIGS. 10A-10E show the structural characterization and activity ofADC-X. FIG. 10A shows the chemical structure of ADC-X. FIG. 10B showsthe potency of IFN stimulatory activity in a luciferase reporter assayusing mouse RAW-Lucia ISG cells. FIGS. 10C and 10D show tumorprogression and survival, respectively, in EGFR-expressing B16F10 tumorbearing C57BL6 mice under indicated treatments. I.p. injections occurredon days 6, 10, and 13. The comparator anti-HER2 antibody is Ab-C1(trastuzumab), and the comparator anti-PDL1 antibody is Ab-A3. FIG. 10Eshows tumor progression in EGFR-expressing B16F10 tumor bearing C57BL6mice under indicated treatments. I.p. and intratumoral (i.t.) injectionsoccurred on days 7 and 11.

6. DETAILED DESCRIPTION

The present disclosure provides antibody-drug conjugates (ADCs), eachcomprising an antibody, one or more cyclic di-nucleotides (CDNs), andone or more linkers that connect the one or more CDNs to the antibody.The ADCs of the disclosure have the ability to agonize and/or bind STINGand promote an immune response.

6.1. Antibody-Drug Conjugates (ADCs)

In certain embodiments, the ADCs of the present disclosure generallyhave the structure of Formula I:Ab-[-L-(D)_(m)]_(n)  (Formula I)wherein:

“D” represents a CDN (e.g., a CDN as described herein, such as those ofFormula II);

“Ab” represents an antibody or binding fragment thereof which binds atarget antigen;

“L” represents, independently for each occurrence, a linker linking oneor more occurrences of D to Ab;

“m” represents the number of occurrences of D linked to a given linker;and

“n” represents the number of linkers linked to Ab.

In certain embodiments of Formula I, m represents an integer selectedfrom 1 to 10, including 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In someinstances, m ranges from 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7,1 to 8, 1 to 9, or 1 to 10, such as from 1 to 2, 1 to 3, 1 to 4, 1 to 5,or 1 to 6. In other embodiments, Formula I describes the ADCs in amixture of ADCs exhibiting a range of values for m, such that m rangesfrom 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9, or1 to 10, such as from 1 to 2, 1 to 3, 1 to 4, 1 to 5, or 1 to 6. Incertain embodiments, Formula I describes the ADCs in a mixture of ADCssuch that more than 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of theADCs in the mixture have an m value of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.In some embodiments, Formula I describes the ADCs in a mixture of ADCssuch that more than 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of theADCs in the mixture have an m value that ranges from 1 to 2, 1 to 3, 1to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8 1, to 9, or 1 to 10, such as from 1to 2, 1 to 3, 1 to 4, 1 to 5, or 1 to 6.

In other embodiments, Formula I describes the ADCs in a mixture of ADCsand m is replaced by “m_(ave)”, which represents the average of m valuesfor the mixture, i.e., the average number of CDNs linked to a givenlinker (L) in the mixture, which can be calculated by dividing the totalnumber of antibody-linked CDNs by the total number of CDN-containinglinkers (L) in the mixture. In such embodiments, m_(ave) represents aninteger or non-integer value ranging from 1 to 10, such as ranging from1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, or 1 to 9, suchas from 1 to 2, 1 to 3, 1 to 4, 1 to 5, or 1 to 6.

In some embodiments of Formula I, n represents an integer selected from1 to 20, including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, or 20. In some instances, n ranges from 1 to 2, 1 to 3,1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9, 1 to 10, 1 to 11, 1 to12, 1 to 13, 1 to 14, 1 to 15, 1 to 16, 1 to 17, 1 to 18, 1 to 19, or 1to 20, such as from 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, or 1to 8. In other embodiments, Formula I describes the ADCs in a mixture ofADCs exhibiting a range of values for n, such that n ranges from 1 to 2,1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9, 1 to 10, 1 to11, 1 to 12, 1 to 13, 1 to 14, 1 to 15, 1 to 16, 1 to 17, 1 to 18, 1 to19, or 1 to 20, such as from 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1to 7, 1 to 8, 1 to 9, or 1 to 10. In certain embodiments, Formula Idescribes the ADCs in a mixture of ADCs such that more than 60%, 65%,70%, 75%, 80%, 85%, 90%, or 95% of the ADCs in the mixture have an nvalue of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, or 20. In some embodiments, Formula I describes the ADCs in amixture of ADCs such that more than 60%, 65%, 70%, 75%, 80%, 85%, 90%,or 95% of the ADCs in the mixture have an n value that ranges from 1 to2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8 1, to 9, 1 to 10, 1 to11, 1 to 12, 1 to 13, 1 to 14, 1 to 15, 1 to 16, 1 to 17, 1 to 18, 1 to19, or 1 to 20, such as from 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1to 7, 1 to 8, 1 to 9, or 1 to 10.

In other embodiments, Formula I describes the ADCs in a mixture of ADCsand n is replaced by “nave”, which represents the average of n valuesfor the mixture, i.e., the average number of linkers (L) linked to agiven antibody (Ab) in the mixture, which can be calculated by dividingthe total number of antibody-linked linkers (L) by the total number oflinker-containing antibodies (Ab) in the mixture. In such embodiments,nave represents an integer or non-integer value ranging from 1 to 20,such as ranging from 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1to 8, 1 to 9, 1 to 10, 1 to 11, 1 to 12, 1 to 13, 1 to 14, 1 to 15, 1 to16, 1 to 17, 1 to 18, 1 to 19, or 1 to 20, such as from 1 to 2, 1 to 3,1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9, or 1 to 10.

In certain embodiments, m is 1, in which case the ADC has a 1:1 ratio oflinker to CDN and can be represented by Formula Ta:Ab-[-L-D]_(n)  (Formula Ia)wherein:

“D” represents a CDN (e.g., a CDN as described herein, such as those ofFormula II);

“Ab” represents an antibody or binding fragment thereof which binds atarget antigen;

“L” represents, independently for each occurrence, a linker linking oneor more occurrences of D to Ab; and

“n” represents the number of occurrences of D linked to Ab via thelinker (L).

In some embodiments of Formula Ta, n represents an integer selected from1 to 20, including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, or 20. In some instances, n ranges from 1 to 2, 1 to 3,1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9, 1 to 10, 1 to 11, 1 to12, 1 to 13, 1 to 14, 1 to 15, 1 to 16, 1 to 17, 1 to 18, 1 to 19, or 1to 20, such as from 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, or 1to 8. In other embodiments, Formula Ta describes the ADCs in a mixtureof ADCs exhibiting a range of values for n, such that n ranges from 1 to2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9, 1 to 10, 1 to11, 1 to 12, 1 to 13, 1 to 14, 1 to 15, 1 to 16, 1 to 17, 1 to 18, 1 to19, or 1 to 20, such as from 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1to 7, or 1 to 8. In certain embodiments, Formula Ta describes the ADCsin a mixture of ADCs such that more than 60%, 65%, 70%, 75%, 80%, 85%,90%, or 95% of the ADCs in the mixture have an n value of 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In someembodiments, Formula Ta describes the ADCs in a mixture of ADCs suchthat more than 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the ADCs inthe mixture have an n value that ranges from 1 to 2, 1 to 3, 1 to 4, 1to 5, 1 to 6, 1 to 7, 1 to 8 1, to 9, 1 to 10, 1 to 11, 1 to 12, 1 to13, 1 to 14, 1 to 15, 1 to 16, 1 to 17, 1 to 18, 1 to 19, or 1 to 20,such as from 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, or 1 to 8.

In other embodiments, Formula Ta describes the ADCs in a mixture of ADCsand n is replaced by “nave”, which represents the average of n valuesfor the mixture, i.e., the average number of linkers (L) linked to agiven antibody (Ab) in the mixture, which can be calculated by dividingthe total number of antibody-linked linkers (L) by the total number oflinker-containing antibodies (Ab) in the mixture. In such embodiments,nave represents an integer or non-integer value ranging from 1 to 20,such as ranging from 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1to 8, 1 to 9, 1 to 10, 1 to 11, 1 to 12, 1 to 13, 1 to 14, 1 to 15, 1 to16, 1 to 17, 1 to 18, 1 to 19, or 1 to 20, such as from 1 to 2, 1 to 3,1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9, or 1 to 10.

The ADCs disclosed herein are “modular” in nature in that each has theabove modular components Ab, L, and D. Throughout the presentdisclosure, various specific non-limiting embodiments and examples ofthese modular components are described. It is intended that the modulesof all of the specific embodiments described may be combined with eachother as though each specific combination were explicitly describedindividually.

It will also be appreciated by skilled artisans that the various ADCsdescribed herein may be in the form of salts, and in some specificembodiments, pharmaceutically acceptable salts.

6.2. Cyclic Di-Nucleotides (CDNs)

The CDNs used herein comprise two nucleosides joined by two bridgegroups. In certain embodiments, the CDNs are 2′3′-CDNs, meaning thateach nucleoside includes a cyclic 5-carbon sugar (a pentose), whereinthe first nucleoside is linked at the 2′-position of its sugar to the5′-position of the second nucleoside's sugar, e.g., by an interveningbridge group, to form a 2′-5′ linkage, and the second nucleoside islinked at the 3′-position of its sugar to the 5′-position of the firstnucleoside's sugar, e.g., by an intervening bridge group, to form a3′-5′ linkage. Examples of suitable 2′3′-CDNs include 2′3′-cGAMP andanalogs or derivatives thereof, including pharmaceutically acceptablesalts. CDNs of Formula II below are 2′3′-CDNs.

In other embodiments, the CDNs are 3′3′-CDNs, wherein the firstnucleotide is linked to the second nucleotide, e.g., by an interveningbridge group, by a 3′-5′ linkage in analogous fashion as describedabove, and the second nucleotide is linked to the first nucleotide,e.g., by an intervening bridge group, also by a 3′-5′ linkage inanalogous fashion as described above. Examples of suitable 3′3′-CDNsinclude 3′3′-cGAMP and analogs or derivatives thereof, includingpharmaceutically acceptable salts.

In other embodiments, the CDNs are 2′2′-CDNs, wherein the firstnucleotide is linked to the second nucleotide, e.g., by an interveningbridge group, by a 2′-5′ linkage in analogous fashion as describedabove, and the second nucleotide is linked to the first nucleotide,e.g., by an intervening bridge group, also by a 2′-5′ linkage inanalogous fashion as described above. Examples of suitable 2′2′-CDNsinclude 2′2′-cGAMP and analogs or derivatives thereof, includingpharmaceutically acceptable salts.

In other embodiments, the CDNs are 3′2′-CDNs, wherein the firstnucleotide is linked to the second nucleotide, e.g., by an interveningbridge group, by a 3′-5′ linkage in analogous fashion as describedabove, and the second nucleotide is linked to the first nucleotide,e.g., by an intervening bridge group, by a 2′-5′ linkage in analogousfashion as described above. Examples of suitable 3′2′-CDNs include3′2′-cGAMP and analogs or derivatives thereof, includingpharmaceutically acceptable salts.

6.2.1. Nucleosides

In some instances, each nucleoside of the CDN includes a nucleobase thatcan be, independently from the other, a pyrimidine base or a purinebase. For instance, each nucleobase can be a canonical nucleobase (suchas adenine, guanine, thymine, uracil, or cytosine) or a non-canonical,modified, non-natural nucleobase (such as xanthine, hypoxanthine,7-methylguanine, 5,6-dihydrouracil, 5-methylcytosine,5-hydroxymethylcytosine, 1-methylcytosine, 2,6-diaminopurine,6,8-diaminopurine, 2-aminoimidazo[1,2a][1,3,5]triazin-4(1H)-one,6-amino-5-nitropyridin-2-one, iso-guanine, iso-cytosine,5-(2,4-diaminopyrimindine), 4-thiouracil, pseudouracil, etc.). Suitablenucleobases include those described below for variables B¹ and B².

In certain instances, the CDN comprises two nucleosides that comprisetwo pyrimidine bases or two purine bases, or one pyrimidine base and onepurine base. In some embodiments, the two nucleosides comprise twopurine bases, such as an adenine and a guanine, two adenines, or twoguanines, particularly an adenine and a guanine or two adenines. It isunderstood that recitation of the indefinite article “a” or “an” beforea particular nucleobase or other defined chemical structure (e.g., “anadenine”) indicates that both the canonical nucleobase and modifiedvariants thereof are contemplated.

In some embodiments, the CDN comprises two nucleosides that each includea cyclic 5-carbon sugar (a pentose), such as D- or L-ribose ordeoxyribose, D- or L-arabinose, D- or L-lyxose, D- or L-xylose, ormodified forms thereof. In certain instances, one of the pentoses of theCDN (e.g., a ribose) connects to the linker (L) at the 3′-position ofthe sugar, such as via a C₁₋₆alkyl group (e.g., a C₂₋₆alkyl group) atthe 3′ position, the C₁₋₆alkyl group being optionally substituted with ahydroxyl, thiol, amino, C₁₋₆alkylamino, or -PEG-OH group; with ahydroxyl, thiol, amino, or C₁₋₆alkylamino group; with a thiol, amino, orC₁₋₆alkylamino group; or with a thiol or amino group. In otherembodiments, one of the pentoses of the CDN (e.g., a ribose) connects tothe linker (L) at the 2′-position of the sugar, such as via a C₁₋₆alkylgroup (e.g., a C₂₋₆alkyl group) at the 2′ position, the C₁₋₆alkyl groupbeing optionally substituted with a hydroxyl, thiol, amino,C₁₋₆alkylamino, or -PEG-OH group; with a hydroxyl, thiol, amino, orC₁₋₆alkylamino group; with a thiol, amino, or C₁₋₆alkylamino group; orwith a thiol or amino group. In some embodiments, the linker (L) isconnected to the CDN by substituting for a proton of the hydroxyl,thiol, amino, C₁₋₆alkylamino, or -PEG-OH group.

The following descriptions of the group at the 3′-position of thepentose can also apply to the group at the 2′-position of the pentose.

In certain instances, the group at the 3′-position of the pentose (e.g.,a ribose) is a C₁₋₆alkyl group, such as a C₂₋₆alkyl group, substitutedwith a hydroxyl, such as -ethylene-OH, -propylene-OH, -butylene-OH, or-pentylene-OH. In some embodiments, the linker (L) is connected bysubstituting for the proton of the hydroxyl in one of the above groups.

In some instances, the group at the 3′-position of the pentose (e.g., aribose) is a C₁₋₆alkyl group, such as a C₂₋₆alkyl group, substitutedwith a thiol, such as -ethylene-SH, -propylene-SH, -butylene-SH, or-pentylene-SH. In some embodiments, the linker (L) is connected bysubstituting for the proton of the thiol in one of the above groups.

In certain instances, the group at the 3′-position of the pentose (e.g.,a ribose) is a C₁₋₆alkyl group, such as a C₂₋₆alkyl group, substitutedwith an amino, such as -ethylene-NH₂, -propylene-NH₂, -butylene-NH₂, or-pentylene-NH₂. In some embodiments, the linker (L) is connected bysubstituting for a proton of the amino in one of the above groups.

In some embodiments, the group at the 3′-position of the pentose (e.g.,a ribose) is a C₁₋₆alkyl group, such as a C₂₋₆alkyl group, substitutedwith a C₁₋₆alkylamino, such as -ethylene-N(C₁₋₆alkyl)H,-propylene-N(C₁₋₆alkyl)H, -butylene-N(C₁₋₆alkyl)H, or-pentylene-N(C₁₋₆alkyl)H. In some embodiments, the linker (L) isconnected by substituting for a proton of the C₁₋₆alkylamino in one ofthe above groups.

In certain embodiments, the group at the 3′-position of the pentose(e.g., a ribose) is a C₁₋₆alkyl group, such as a C₂₋₆alkyl group,substituted with a -PEG-OH group, such as -ethylene-PEG-OH,-propylene-PEG-OH, -butylene-PEG-OH, or -pentylene-PEG-OH. In someembodiments, the linker (L) is connected by substituting for a proton ofthe terminal hydroxyl of the -PEG-OH group. It is understood that “PEG”refers to the polymer polyethylene glycol, polyethylene oxide, orpolyoxyethylene and having the repeating structure —(O—CH₂—CH₂)_(x)— andaverage molecular weights ranging from 200 to 10000 g/mol, such as from200, 400, 800, 1000, 2000, or 4000 to 5000, 6000 8000 or 10000 g/mol,including from 400 to 8,000 g/mol, 400 to 2000 g/mol, 5000 to 10000g/mol, 1000 to 4000 g/mol, 1000 to 6000 g/mol, or 2000 to 6000 g/mol,including less than 4000, 5000, or 6000 g/mol.

In some instances, the group at the 3′-position of the pentose (e.g., aribose) is a C₂₋₃alkyl group substituted with hydroxyl, thiol, amino, aC₁₋₆alkylamino, or -PEG-OH group, such as -ethylene-OH, -propylene-OH,-ethylene-SH, -propylene-SH, -ethylene-NH₂, -propylene-NH₂,-ethylene-N(C₁₋₆alkyl)H, -propylene-N(C₁₋₆alkyl)H, -ethylene-PEG-OH, or-propylene-PEG-OH. In some embodiments, the linker (L) is connected bysubstituting for a proton of the hydroxyl, thiol, amino, orC₁₋₆alkylamino or -PEG-OH group in one of the above groups.

In some instances, the group at the 3′-position of the pentose (e.g., aribose) is a C₂alkyl group (an ethyl group) substituted with hydroxyl,thiol, amino, a C₁₋₆alkylamino, or -PEG-OH group, such as -ethylene-OH,-ethylene-SH, -ethylene-NH₂, -ethylene-N(C₁₋₆alkyl)H, or-ethylene-PEG-OH. In some embodiments, the linker (L) is connected bysubstituting for a proton of the hydroxyl, thiol, amino, orC₁₋₆alkylamino or -PEG-OH group in one of the above groups.

In some instances, the group at the 3′-position of the pentose (e.g., aribose) is a C₃alkyl group (a propyl group) substituted with hydroxyl,thiol, amino, a C₁₋₆alkylamino, or -PEG-OH group, such as -propylene-OH,-propylene-SH, -propylene-NH₂, -propylene-N(C₁₋₆alkyl)H, or-propylene-PEG-OH. In some embodiments, the linker (L) is connected bysubstituting for a proton of the hydroxyl, thiol, amino, orC₁₋₆alkylamino or -PEG-OH group in one of the above groups.

In certain embodiments, one of the pentoses of the CDN (e.g., a ribose)connects to the linker (L) at the 3′-position of the sugar via asubstituted methyl group at the 3′ position, the methyl group beingsubstituted, for example, with a hydroxyl, thiol, amino, C₁₋₆alkylamino,or -PEG-OH group; with a hydroxyl, thiol, amino, or C₁₋₆alkylaminogroup; with a thiol, amino, or C₁₋₆alkylamino group; with a thiol oramino group; or with a thiol group. In some embodiments, the linker (L)is connected to the CDN by substituting for a proton of the hydroxyl,thiol, amino, C₁₋₆alkylamino, or -PEG-OH group.

In certain embodiments, the CDN comprises two nucleosides that eachinclude a ribose, wherein one of the riboses connects to the linker (L)via a C₂₋₆alkyl group (such as a C₂alkyl, C₃alkyl, or C₂₋₃alkyl group)at the 3′-position of the ribose ring, the C₂₋₆alkyl group beingoptionally substituted with a hydroxyl, thiol, amino, C₁₋₆alkylamino, or-PEG-OH group; with a hydroxyl, thiol, amino, or C₁₋₆alkylamino group;with a thiol, amino, or C₁₋₆alkylamino group; or with a thiol or aminogroup; wherein the linker (L) is connected to the CDN by substitutingfor a proton of the hydroxyl, thiol, amino, C₁₋₆alkylamino, or -PEG-OHgroup.

In certain embodiments, the CDN comprises two nucleosides that eachinclude a ribose, wherein one of the riboses connects to the linker (L)via a substituted ethyl group at the 3′-position of the ribose ring, theethyl group being substituted, for example, with a hydroxyl, thiol,amino, C₁₋₆alkylamino, or -PEG-OH group; with a hydroxyl, thiol, amino,or C₁₋₆alkylamino group; with a thiol, amino, or C₁₋₆alkylamino group;or with a thiol or amino group; wherein the linker (L) is connected tothe CDN by substituting for a proton of the hydroxyl, thiol, amino,C₁₋₆alkylamino, or -PEG-OH group.

In certain embodiments, the CDN comprises two nucleosides that eachinclude a ribose, wherein one of the riboses connects to the linker (L)via a substituted ethyl group at the 3′-position of the ribose ringselected from —CH₂CH₂—OH, —CH₂CH₂—SH, and —CH₂CH₂—NH₂, particularly from—CH₂CH₂—SH and —CH₂CH₂—NH₂; wherein the linker (L) is connected to theCDN by substituting for a proton of the hydroxyl, thiol, or amino group.

6.2.2. Bridge Groups

Due to their cyclic structure, CDNs include two bridge groups that jointhe nucleosides described above to form the CDN macrocycle. In certainembodiments, the bridge groups, independently, include 2 to 5 atoms inthe bridge between the two sugars of the nucleosides, such as 3 atoms.For instance, —O—P(═O)(OH)—O— may be a bridge group, where it isunderstood that the sugars are bonded at the terminal oxygen atoms, andthere are three atoms in the bridge. The bridge groups, independently,may include only heteroatoms in the bridge, both heteroatoms and carbonatoms in the bridge, or only carbon atoms in the bridge.

In certain instances the bridge groups are divalent phosphate orthiophosphate groups or modified variants thereof, e.g., —O—P(═O)(OH)—O—or —O—P(═O)(SH)—O—. For example, the two bridge groups can beindependently selected from —O—P(O)R^(p)—O—, —O—P(S)R^(p)—O—,—O—P(O)R^(p)—S—, —O—P(S)R^(p)—S—, —S—P(O)R^(p)—O—, —S—P(S)R^(p)—O—,—S—P(O)R^(p)—S—, or —S—P(S)R^(p)—S—, wherein R^(p) is defined furtherbelow. In some embodiments, R^(p) in the above bridge groupsindependently for each occurrence selected from hydroxyl or thiol.

It is understood that when both bridge groups are phosphate groups ormodified variants thereof, then each bridge group in combination with anucleoside described above represents a nucleotide, with both sets ofbridge groups and nucleosides providing a cyclic dinucleotide.Nevertheless, CDNs disclose herein are not necessarily limited to bridgegroups that are phosphate groups.

6.2.3. Specific CDNs

The present disclosure provides CDNs (D) that can be administered bythemselves or as part of the ADC of Formula I. In certain instances, theCDN has the structure of Formula II below. It is understood thatreference to Formula I also includes reference to sub Formula Ia.Likewise, reference to Formula II also includes references to its subformulas, such as Formula IIa, IIb, etc. Formula II has the structure:

wherein

R¹ is C₁₋₆alkyl, such as C₂₋₆alkyl or C₂₋₃alkyl, substituted with ahydroxyl, thiol, amino, C₁₋₆alkylamino, or a -PEG-OH group;

R³ and R⁴ are independently hydrogen, halogen, C₁₋₆alkyl, C₂₋₆alkenyl,or C₂₋₆alkynyl, wherein C₁₋₆alkyl, C₂₋₆alkenyl, and C₂₋₆alkynyl are,independently, optionally substituted with one or more groups selectedfrom halogen, thiol, hydroxyl, carboxyl, C₁₋₆alkoxy, C₁₋₆hydroxyalkoxy,—OC(O)C₁₋₆alkyl, —N(H)C(O)C₁₋₆alkyl, —N(C₁₋₃alkyl)C(O)C₁₋₆alkyl, amino,C₁₋₆alkylamino, di(C₁₋₆alkyl)amino, oxo, and azido;

R², R⁵, and R⁶ are independently hydrogen, halogen, hydroxyl, azido,amino, C₁₋₆alkylamino, di(C₁₋₆alkyl)amino, C₁₋₆alkyl, C₁₋₆alkoxy,C₂₋₆alkenyl, C₃₋₆alkenyl-O—, C₂₋₆alkynyl, or C₃₋₆alkynyl-O—, whereinC₁₋₆alkyl, C₁₋₆alkoxy, C₂₋₆alkenyl, C₃₋₆alkenyl-O—, C₂₋₆alkynyl, andC₃₋₆alkynyl-O—, are, independently, optionally substituted with one ormore groups selected from halogen, thiol, hydroxyl, carboxyl,C₁₋₆alkoxy, C₁₋₆hydroxyalkoxy, —OC(O)C₁₋₆alkyl, —N(H)C(O)C₁₋₆alkyl,—N(C₁₋₃alkyl)C(O)C₁₋₆alkyl, amino, C₁₋₆alkylamino, di(C₁₋₆alkyl)amino,oxo, and azido; or R⁶ and R⁵ together are ═CH₂; or R⁶ and R⁴ togetherform a bridge across the ring containing V² selected from ethylene,—O—CH₂—, and —NH—CH₂—;

V¹ and V² are independently O, S, or CH₂;

BG¹, starting from the carbon in the ring containing VI, and BG²,starting from the carbon in the ring containing V², are independently—O—P(O)R^(p)—O—, —O—P(S)R^(p)—O—, —O—P(O)R^(p)—S—, —O—P(S)R^(p)—S—,—S—P(O)R^(p)—O—, —S—P(S)R^(p)—O—, —S—P(O)R^(p)—S—, —S—P(S)R^(p)—S—,—NH—P(O)R^(p)—O—, —O—P(O)R^(p)—NH—, —NH—P(S)R^(p)—O—, —O—P(S)R^(p)—NH—,or —NH—SO₂—NH—; wherein

-   -   R^(p) is, independently for each occurrence, hydroxyl, thiol,        C₁₋₆alkyl, C₁₋₆alkoxy, C₃₋₆alkenyl-O—, C₃₋₆alkynyl-O—, -PEG-OH,        borano (—BH₃ ⁻), or —NR′R″, wherein C₁₋₆alkyl, C₁₋₆alkoxy,        C₃₋₆alkenyl-O—, and C₃₋₆alkynyl-O—, are, independently,        optionally substituted with one or more groups selected from        halogen, thiol, hydroxyl, carboxyl, C₁₋₆alkoxy,        C₁₋₆hydroxyalkoxy, —OC(O)C₁₋₆alkyl, —N(H)C(O)C₁₋₆alkyl,        —N(C₁₋₃alkyl)C(O)C₁₋₆alkyl, amino, C₁₋₆alkylamino,        di(C₁₋₆alkyl)amino, oxo, and azido; and    -   R′ and R″ are independently hydrogen or C₁₋₆alkyl optionally        substituted with one or more groups selected from halogen,        thiol, hydroxyl, carboxyl, C₁₋₆alkoxy, C₁₋₆hydroxyalkoxy,        —OC(O)C₁₋₆alkyl, —N(H)C(O)C₁₋₆alkyl, —N(C₁₋₃alkyl)C(O)C₁₋₆alkyl,        amino, C₁₋₆alkylamino, di(C₁₋₆alkyl)amino, oxo, and azido; or R′        and R″ on the same nitrogen together form a C₃₋₅heterocyclic        ring;

R^(a1), R^(b1), R^(a2), and R^(b2) are independently hydrogen orC₁₋₃alkyl; and

B¹ and B² are independently selected from:

wherein

-   -   V³ is O or S, particularly O;    -   Z¹, Z², Z³, Z⁴, Z⁵, and Z⁶ are, independently for each        occurrence, CR^(z) or N;    -   Z^(a) is O (except when Z⁵ is N) or NR′; wherein        -   R^(z) is, independently for each occurrence, hydrogen,            halogen, azido, amino, C₁₋₆alkylamino, di(C₁₋₆alkyl)amino,            C₁₋₆alkyl, C₁₋₆alkoxy, C₂₋₆alkenyl, C₃₋₆alkenyl-O—,            C₂₋₆alkynyl, C₃₋₆alkynyl-O—, —NO₂, —CN, —C(O)C₁₋₆alkyl,            —CO₂H, —CO₂C₁₋₆alkyl, —S(O)C₁₋₆alkyl, —S(O)₂C₁₋₆alkyl,            —C(O)NR′, —C(O)NR′R″, —SO₂NR′R″, —OC(O)C₁₋₆alkyl,            —NR′C(O)C₁₋₆alkyl, —N(R′)C(O)NR′R″, —N(R′)SO₂NR′R″,            —N(R)SO₂C₁₋₆alkyl, or —OC(O)NR′R″, wherein        -   C₁₋₆alkyl, C₁₋₆alkoxy, C₂₋₆alkenyl, C₃₋₆alkenyl-O—,            C₂₋₆alkynyl, and C₃₋₆alkynyl-O—, are, independently for each            occurrence, optionally substituted with one or more groups            selected from halogen, thiol, hydroxyl, carboxyl,            C₁₋₆alkoxy, C₁₋₆hydroxyalkoxy, —OC(O)C₁₋₆alkyl,            —N(H)C(O)C₁₋₆alkyl, —N(C₁₋₃alkyl)C(O)C₁₋₆alkyl, amino,            C₁₋₆alkylamino, di(C₁₋₆alkyl)amino, oxo, and azido; and        -   R′ and R″ are, independently for each occurrence, hydrogen            or C₁₋₆alkyl optionally substituted with one or more groups            selected from halogen, thiol, hydroxyl, carboxyl,            C₁₋₆alkoxy, C₁₋₆hydroxyalkoxy, —OC(O)C₁₋₆alkyl,            —N(H)C(O)C₁₋₆alkyl, —N(C₁₋₃alkyl)C(O)C₁₋₆alkyl, amino,            C₁₋₆alkylamino, di(C₁₋₆alkyl)amino, oxo, and azido; or R′            and R″ on the same nitrogen together form a C₃₋₅heterocyclic            ring;            or a pharmaceutically acceptable salt thereof.

In some instances, when the CDN (D) of Formula II is covalently bound tolinker (L) in an ADC of Formula I or Ta, the CDN is covalently bound tothe linker at the hydroxyl, thiol, amino, C₁₋₆alkylamino, or -PEG-OHgroup of the R¹ position.

In certain embodiments, R¹ is C₂₋₆alkyl substituted with a hydroxyl,thiol, amino, C₁₋₆alkylamino, or a -PEG-OH group. In some embodiments,R¹ is C₂₋₆alkyl substituted with a hydroxyl, thiol, amino, orC₁₋₆alkylamino. In certain embodiments, R¹ is C₂₋₆alkyl substituted witha thiol, amino, or C₁₋₆alkylamino. In some embodiments, R¹ is C₂₋₆alkylsubstituted with a thiol or amino. In certain embodiments, R¹ isC₂₋₆alkyl substituted with a thiol. In some embodiments, R¹ is C₂₋₆alkylsubstituted with an amino. For any of these embodiments, R¹ can beC₂₋₄alkyl, such as C₂₋₃alkyl, substituted with hydroxyl, thiol, amino,or C₁₋₆alkylamino, such as ethyl substituted with hydroxyl, thiol,amino, or C₁₋₆alkylamino.

In some embodiments, R¹ is C₂₋₄alkyl substituted with a hydroxyl group.In some such embodiments, R¹ is a C₂alkyl substituted with a hydroxyl.In other such embodiments, R¹ is a C₃alkyl substituted with a hydroxyl.In yet other such embodiments, R¹ is a C₄alkyl substituted with ahydroxyl.

In some embodiments, R¹ is C₂₋₄alkyl substituted with an amino (—NH₂)group. In some such embodiments, R¹ is a C₂alkyl substituted with anamino group. In other such embodiments, R¹ is a C₃alkyl substituted withan amino group. In yet other such embodiments, R¹ is a C₄alkylsubstituted with an amino group.

In some embodiments, R¹ is C₂₋₄alkyl substituted with a thiol (—SH)group. In some such embodiments, R¹ is a C₂alkyl substituted with athiol group. In other such embodiments, R¹ is a C₃alkyl substituted witha thiol group. In yet other such embodiments, R¹ is a C₄alkylsubstituted with a thiol group.

In some embodiments, R¹ is a C₂₋₄alkyl substituted with a C₁₋₆alkylaminogroup. In some such embodiments, R¹ is a C₂alkyl substituted with amethylamino group. In other such embodiments, R¹ is a C₃alkylsubstituted with a methylamino group. In yet other such embodiments, R¹is a C₃alkyl substituted with a methylamino group.

In some embodiments, R³ and R⁴ are independently hydrogen, halogen,C₁₋₆alkyl, C₂₋₆alkenyl, or C₂₋₆alkynyl, wherein C₁₋₆alkyl, C₂₋₆alkenyl,and C₂₋₆alkynyl are unsubstituted. In certain embodiments, R³ and R⁴ areindependently hydrogen, C₁₋₆alkyl, or C₂₋₆alkynyl, wherein C₁₋₆alkyl andC₂₋₆alkynyl are unsubstituted.

In some embodiments, one of R³ and R⁴ is hydrogen and the other ishalogen, C₁₋₆alkyl, C₂₋₆alkenyl, or C₂₋₆alkynyl, wherein C₁₋₆alkyl,C₂₋₆alkenyl, and C₂₋₆alkynyl are, independently, optionally substitutedwith one or more groups selected from halogen, thiol, hydroxyl,carboxyl, C₁₋₆alkoxy, C₁₋₆hydroxyalkoxy, —OC(O)C₁₋₆alkyl,—N(H)C(O)C₁₋₆alkyl, —N(C₁₋₃alkyl)C(O)C₁₋₆alkyl, amino, C₁₋₆alkylamino,di(C₁₋₆alkyl)amino, oxo, and azido. In some embodiments, one of R³ andR⁴ is hydrogen and the other is halogen, C₁₋₆alkyl, C₂₋₆alkenyl, orC₂₋₆alkynyl, wherein C₁₋₆alkyl, C₂₋₆alkenyl, and C₂₋₆alkynyl areunsubstituted. In certain embodiments, one of R³ and R⁴ is hydrogen andthe other is C₁₋₆alkyl or C₂₋₆alkynyl, wherein C₁₋₆alkyl and C₂₋₆alkynylare unsubstituted. In some embodiments, both R³ and R⁴ are hydrogen.

In certain embodiments, R² is hydrogen, halogen, hydroxyl, azido, amino,C₁₋₆alkyl, or C₁₋₆alkoxy, wherein C₁₋₆alkyl and C₁₋₆alkoxy are,independently, optionally substituted with one or more groups selectedfrom halogen, thiol, hydroxyl, carboxyl, C₁₋₆alkoxy, C₁₋₆hydroxyalkoxy,—OC(O)C₁₋₆alkyl, —N(H)C(O)C₁₋₆alkyl, —N(C₁₋₃alkyl)C(O)C₁₋₆alkyl, amino,C₁₋₆alkylamino, di(C₁₋₆alkyl)amino, oxo, and azido. In some embodiments,R² is hydrogen, halogen, hydroxyl, azido, amino, C₁₋₆alkyl, orC₁₋₆alkoxy, wherein C₁₋₆alkyl and C₁₋₆alkoxy are unsubstituted. Incertain embodiments, R² is hydrogen or halogen, such as fluorine. Ininstances, R² is hydrogen.

In some embodiments, R⁵ and R⁶ are independently hydrogen, halogen,hydroxyl, azido, amino, C₁₋₆alkyl, or C₁₋₆alkoxy, wherein C₁₋₆alkyl andC₁₋₆alkoxy are, independently, optionally substituted with one or moregroups selected from halogen, thiol, hydroxyl, carboxyl, C₁₋₆alkoxy,C₁₋₆hydroxyalkoxy, —OC(O)C₁₋₆alkyl, —N(H)C(O)C₁₋₆alkyl,—N(C₁₋₃alkyl)C(O)C₁₋₆alkyl, amino, C₁₋₆alkylamino, di(C₁₋₆alkyl)amino,oxo, and azido. In certain embodiments, R⁵ and R⁶ are independentlyhydrogen, halogen, hydroxyl, azido, amino, C₁₋₆alkyl, or C₁₋₆alkoxy,wherein C₁₋₆alkyl and C₁₋₆alkoxy are unsubstituted. In some embodiments,R⁵ and R⁶ are independently hydrogen, halogen, or hydroxyl. In certainembodiments, R⁵ and R⁶ together are ═CH₂.

In certain embodiments, R⁵ is hydrogen and R⁶ is hydrogen, halogen,hydroxyl, azido, amino, C₁₋₆alkylamino, di(C₁₋₆alkyl)amino, C₁₋₆alkyl,C₁₋₆alkoxy, C₂₋₆alkenyl, C₃₋₆alkenyl-O—, C₂₋₆alkynyl, or C₃₋₆alkynyl-O—,wherein C₁₋₆alkyl, C₁₋₆alkoxy, C₂₋₆alkenyl, C₃₋₆alkenyl-O—, C₂₋₆alkynyl,and C₃₋₆alkynyl-O—, are, independently, optionally substituted with oneor more groups selected from halogen, thiol, hydroxyl, carboxyl,C₁₋₆alkoxy, C₁₋₆hydroxyalkoxy, —OC(O)C₁₋₆alkyl, —N(H)C(O)C₁₋₆alkyl,—N(C₁₋₃alkyl)C(O)C₁₋₆alkyl, amino, C₁₋₆alkylamino, di(C₁₋₆alkyl)amino,oxo, and azido. In some embodiments, R⁵ is hydrogen and R⁶ is hydrogen,halogen, hydroxyl, azido, amino, C₁₋₆alkyl, or C₁₋₆alkoxy, whereinC₁₋₆alkyl and C₁₋₆alkoxy are, independently, optionally substituted withone or more groups selected from halogen, thiol, hydroxyl, carboxyl,C₁₋₆alkoxy, C₁₋₆hydroxyalkoxy, —OC(O)C₁₋₆alkyl, —N(H)C(O)C₁₋₆alkyl,—N(C₁₋₃alkyl)C(O)C₁₋₆alkyl, amino, C₁₋₆alkylamino, di(C₁₋₆alkyl)amino,oxo, and azido. In certain embodiments, R⁵ is hydrogen and R⁶ ishydrogen, halogen, hydroxyl, azido, amino, C₁₋₆alkyl, or C₁₋₆alkoxy,wherein C₁₋₆alkyl and C₁₋₆alkoxy are unsubstituted. In some embodiments,R⁵ is hydrogen and R⁶ is hydrogen, halogen (such as fluorine orchlorine), hydroxyl, or unsubstituted C₁₋₆alkoxy (such as methoxy). Insome embodiments, R⁵ is hydrogen and R⁶ is halogen, such as fluorine orchlorine. In certain instances, R⁵ is hydrogen and R⁶ is hydroxyl. Inother embodiments, R⁵ is hydrogen and R⁶ is unsubstituted C₁₋₆alkoxy,such as methoxy.

In certain embodiments, R⁶ is hydrogen and R⁵ is hydrogen, halogen,hydroxyl, azido, amino, C₁₋₆alkylamino, di(C₁₋₆alkyl)amino, C₁₋₆alkyl,C₁₋₆alkoxy, C₂₋₆alkenyl, C₃₋₆alkenyl-O—, C₂₋₆alkynyl, or C₃₋₆alkynyl-O—,wherein C₁₋₆alkyl, C₁₋₆alkoxy, C₂₋₆alkenyl, C₃₋₆alkenyl-O—, C₂₋₆alkynyl,and C₃₋₆alkynyl-O—, are, independently, optionally substituted with oneor more groups selected from halogen, thiol, hydroxyl, carboxyl,C₁₋₆alkoxy, C₁₋₆hydroxyalkoxy, —OC(O)C₁₋₆alkyl, —N(H)C(O)C₁₋₆alkyl,—N(C₁₋₃alkyl)C(O)C₁₋₆alkyl, amino, C₁₋₆alkylamino, di(C₁₋₆alkyl)amino,oxo, and azido. In some embodiments, R⁶ is hydrogen and R⁵ is hydrogen,halogen, hydroxyl, azido, amino, C₁₋₆alkyl, or C₁₋₆alkoxy, whereinC₁₋₆alkyl and C₁₋₆alkoxy are, independently, optionally substituted withone or more groups selected from halogen, thiol, hydroxyl, carboxyl,C₁₋₆alkoxy, C₁₋₆hydroxyalkoxy, —OC(O)C₁₋₆alkyl, —N(H)C(O)C₁₋₆alkyl,—N(C₁₋₃alkyl)C(O)C₁₋₆alkyl, amino, C₁₋₆alkylamino, di(C₁₋₆alkyl)amino,oxo, and azido. In certain embodiments, R⁶ is hydrogen and R⁵ ishydrogen, halogen, hydroxyl, azido, amino, C₁₋₆alkyl, or C₁₋₆alkoxy,wherein C₁₋₆alkyl and C₁₋₆alkoxy are unsubstituted. In some embodiments,R⁶ is hydrogen and R⁵ is hydrogen, halogen (such as fluorine orchlorine), hydroxyl, or unsubstituted C₁₋₆alkoxy (such as methoxy). Insome embodiments, R⁶ is hydrogen and R⁵ is halogen, such as fluorine orchlorine. In certain instances, R⁶ is hydrogen and R⁵ is hydroxyl. Inother embodiments, R⁵ is hydrogen and R⁶ is unsubstituted C₁₋₆alkoxy,such as methoxy.

In some embodiments, one of R⁵ and R⁶ is hydrogen and the other ishalogen, such as fluorine. In certain embodiments, R⁵ is hydrogen and R⁶is halogen, such as fluorine. In certain embodiments, R⁶ is hydrogen andR⁵ is halogen, such as fluorine.

In certain instances, both R⁵ and R⁶ are hydrogen or halogen, such asboth R⁵ and R⁶ are hydrogen, or both R⁵ and R⁶ are halogen, such asfluorine.

In some embodiments, R⁵ is hydrogen. In other embodiments, R⁵ ishydroxyl. In other embodiments, R⁵ is halogen. For instance, R⁵ can befluorine, bromine, or chlorine.

In some embodiments, R⁶ is hydrogen. In other embodiments, R⁶ ishydroxyl. In other embodiments, R⁶ is methoxy. In other embodiments, R⁶is halogen. For instance, R⁶ can be fluorine, bromine, or chlorine.

In certain embodiments, V¹ and V² are independently O or S. In someembodiments, V¹ and V² are independently O or CH₂. In some embodiments,V¹ and V² are both O. In certain embodiments, V¹ and V² are both S. Incertain embodiments, at least one of VI and V² is O. In someembodiments, at least one of V¹ and V² is S. In certain embodiments, atleast one of V¹ and V² is CH₂.

In some embodiments, BG¹ and BG² are independently —O—P(O)R^(p)—O— or—O—P(S)R^(p)—O—. In certain embodiments, at least one of BG¹ and BG² is—O—P(O)R^(p)—O—. In some embodiments, at least one of BG¹ and BG² is—O—P(S)R^(p)—O—. In certain embodiments, BG¹ is —O—P(O)R^(p)—O— and BG²is —O—P(S)R^(p)—O—. In some embodiments, BG¹ is —O—P(O)R^(p)—O— and BG²is —O—P(S)R^(p)—O—. In certain embodiments, both BG¹ and BG² are—O—P(O)R^(p)—O—.

In certain embodiments, R^(p) is, independently for each occurrence,hydroxyl, thiol, C₁₋₆alkyl, borano (—BH₃ ⁻), or —NR′R″. In someembodiments, R^(p) is, independently for each occurrence, hydroxyl orthiol. In certain embodiments, R^(p) is hydroxyl. In some embodiments,R^(p) is thiol. In some embodiments, one R^(p) is hydroxyl and the otheris thiol.

In some embodiments, both BG¹ and BG² are —O—P(O)R^(p)—O— and R^(p) is,independently for each occurrence, hydroxyl, thiol, C₁₋₆alkyl, borano(—BH₃ ⁻), or —NR′R″. In certain embodiments, both BG¹ and BG² are—O—P(O)R^(p)—O— and R^(p) is, independently for each occurrence,hydroxyl or thiol. In some embodiments, both BG¹ and BG² are—O—P(O)R^(p)—O— and R^(p) is hydroxyl. In other embodiments, both BG¹and BG² are —O—P(O)R^(p)—O— and R^(p) is thiol. In some embodiments, BG¹is —O—P(O)R^(p)—O—, wherein R^(p) is hydroxyl, and BG² is—O—P(O)R^(p)—O—, wherein R^(p) is thiol. In other embodiments, BG¹ is—O—P(O)R^(p)—O—, wherein R^(p) is thiol, and BG² is —O—P(O)R^(p)—O—,wherein R^(p) is hydroxyl.

In certain instances, at least one of BG¹ and BG² is —NH—P(O)R^(p)—O—,—O—P(O)R^(p)—NH—, —NH—P(S)R^(p)—O—, or —O—P(S)R^(p)—NH—. In someinstances, at least one of BG¹ and BG² is —NH—P(O)R^(p)—O—, or—O—P(O)R^(p)—NH—. In certain embodiments, both BG¹ and BG² are—NH—P(O)R^(p)—O— or —O—P(O)R^(p)—NH—. In certain instances, at least oneof BG¹ and BG² is —NH—P(O)R^(p)—O—, —O—P(O)R^(p)—NH—, —NH—P(S)R^(p)—O—,or —O—P(S)R^(p)—NH—; and R^(p) is, independently for each occurrence,hydroxyl or thiol. In some instances, at least one of BG¹ and BG² is—NH—P(O)R^(p)—O—, or —O—P(O)R^(p)—NH—; and R^(p) is, independently foreach occurrence, hydroxyl or thiol. In certain embodiments, both BG¹ andBG² are —NH—P(O)R^(p)—O— or —O—P(O)R^(p)—NH—; and R^(p) is,independently for each occurrence, hydroxyl or thiol. In someembodiments, both BG¹ and BG² are —NH—P(O)R^(p)—O— or —O—P(O)R^(p)—NH—;and R^(p) is hydroxyl. In other embodiments, both BG¹ and BG² are—NH—P(O)R^(p)—O— or —O—P(O)R^(p)—NH—; and R^(p) is thiol.

In certain embodiments, when R^(p) is —NR′R″, R′ and R″ areindependently hydrogen or unsubstituted C₁₋₆alkyl, or R′ and R″ togetheron the same nitrogen form a C₃₋₅heterocyclic ring, such as morpholine,pyrrolidine, or piperazine.

In certain embodiments, B¹ and B² are the same nucleobase. In otherembodiments, B¹ and B² are different nucleobases. In some embodiments,B¹ and B² are both a purine nucleobase. In certain embodiments, B¹ andB² are both a pyrimidine nucleobase. In some embodiments, B¹ is a purinenucleobase, and B² is a pyrimidine nucleobase. In certain embodiments,B¹ is a pyrimidine nucleobase, and B² is a purine nucleobase.

In some instances, B¹ and B² are independently selected from adenine,guanine, thymine, uracil, and cytosine and modified variants of these.In certain embodiments, both B¹ and B² are adenine or a modified variantthereof. In some embodiments, both B¹ and B² are guanine or a modifiedvariant thereof. In certain embodiments, B¹ is guanine or a modifiedvariant thereof, and B² is adenine or a modified variant thereof. Insome embodiments, B¹ is adenine or a modified variant thereof, and B² isguanine or a modified variant thereof.

In some embodiments, B¹ and B² are independently selected from:

wherein

Z¹, Z², Z³, Z⁴, Z⁵, and Z⁶ are, independently for each occurrence,CR^(z) or N;

Z^(a) is O (except when Z⁵ is N) or NR′; wherein

-   -   R^(z) is, independently for each occurrence, hydrogen, halogen,        azido, amino, C₁₋₆alkylamino, di(C₁₋₆alkyl)amino, C₁₋₆alkyl,        C₁₋₆alkoxy, —C(O)C₁₋₆alkyl, —CO₂H, —CO₂C₁₋₆alkyl, —C(O)NR′,        —C(O)NR′R″, —OC(O)C₁₋₆alkyl, —NR′C(O)C₁₋₆alkyl, —N(R′)C(O)NR′R″,        or —OC(O)NR′R″, wherein        -   C₁₋₆alkyl and C₁₋₆alkoxy are, independently for each            occurrence, optionally substituted with one or more groups            selected from halogen, thiol, hydroxyl, carboxyl,            C₁₋₆alkoxy, C₁₋₆hydroxyalkoxy, —OC(O)C₁₋₆alkyl,            —N(H)C(O)C₁₋₆alkyl, —N(C₁₋₃alkyl)C(O)C₁₋₆alkyl, amino,            C₁₋₆alkylamino, di(C₁₋₆alkyl)amino, oxo, and azido; and        -   R′ and R″ are, independently for each occurrence, hydrogen            or C₁₋₆alkyl optionally substituted with one or more groups            selected from halogen, thiol, hydroxyl, carboxyl,            C₁₋₆alkoxy, C₁₋₆hydroxyalkoxy, —OC(O)C₁₋₆alkyl,            —N(H)C(O)C₁₋₆alkyl, —N(C₁₋₃alkyl)C(O)C₁₋₆alkyl, amino,            C₁₋₆alkylamino, di(C₁₋₆alkyl)amino, oxo, and azido; or R′            and R″ on the same nitrogen together form a C₃₋₅heterocyclic            ring.

In some embodiments, for B¹ and B², one or both occurrences of Z¹ areCR^(z) (such as CH or CNH₂). In certain embodiments, one or bothoccurrences of Z⁵ are CR^(z) (such as CH or CNH₂). In some embodiments,one or both occurrences of Z¹ are CR^(z) (such as CH or CNH₂), and oneor both occurrences of Z⁵ are CR^(z) (such as CH or CNH₂). In certainembodiments, both occurrences of Z¹ are CR^(z) (such as CH or CNH₂), andboth occurrences of Z⁵ are CR^(z) (such as CH or CNH₂).

In certain instances, for B¹ and B², Z³ is CR^(z) (such as CH or CNH₂).In certain embodiments, Z³ is CR^(z) (such as CH or CNH₂), and one orboth occurrences of Z¹ are CR^(z)(such as CH or CNH₂). In certainembodiments, Z³ is CR^(z) (such as CH or CNH₂), and one or bothoccurrences of Z⁵ are CR^(z) (such as CH or CNH₂). In certainembodiments, Z³ is CR^(z)(such as CH or CNH₂), both occurrences of Z¹are CR^(z) (such as CH or CNH₂), and both occurrences of Z⁵ are CR^(z)(such as CH or CNH₂).

In some instances, for B¹ and B², Z^(a) is NR′, such as NH orNC₁₋₆alkyl. In other embodiments, Z^(a) is O.

In embodiments instances, one or both occurrences of Z² are N. Incertain embodiments, Z⁴ is N. In some embodiments, one or bothoccurrences of Z² are N, and Z⁴ is N. In certain embodiments, bothoccurrences of Z² are N, and Z⁴ is N.

In embodiments instances, one or both occurrences of Z⁶ are N. Incertain embodiments, Z⁴ is N. In some embodiments, one or bothoccurrences of Z⁶ are N, and Z⁴ is N. In certain embodiments, bothoccurrences of Z⁶ are N, and Z⁴ is N. In some embodiments, one or bothoccurrences of Z⁶ are N, and one or both occurrences of Z² are N. Insome embodiments, both occurrences of Z⁶ are N, and both occurrences ofZ² are N.

In some embodiments, B¹ and B² are independently selected from:

wherein

Z¹, Z³, and Z⁵ are, independently for each occurrence, CR^(z) or N;

Z^(a) is O (except when Z⁵ is N) or NR′; wherein

-   -   R^(z) is, independently for each occurrence, hydrogen, halogen,        azido, amino, C₁₋₆alkylamino, di(C₁₋₆alkyl)amino, C₁₋₆alkyl,        C₁₋₆alkoxy, —C(O)C₁₋₆alkyl, —CO₂H, —CO₂C₁₋₆alkyl, —C(O)NR′,        —C(O)NR′R″, —OC(O)C₁₋₆alkyl, —NR′C(O)C₁₋₆alkyl, —N(R′)C(O)NR′R″,        or —OC(O)NR′R″, wherein        -   C₁₋₆alkyl and C₁₋₆alkoxy are, independently for each            occurrence, optionally substituted with one or more groups            selected from halogen, thiol, hydroxyl, carboxyl,            C₁₋₆alkoxy, C₁₋₆hydroxyalkoxy, —OC(O)C₁₋₆alkyl,            —N(H)C(O)C₁₋₆alkyl, —N(C₁₋₃alkyl)C(O)C₁₋₆alkyl, amino,            C₁₋₆alkylamino, di(C₁₋₆alkyl)amino, oxo, and azido; and        -   R′ and R″ are, independently for each occurrence, hydrogen            or C₁₋₆alkyl optionally substituted with one or more groups            selected from halogen, thiol, hydroxyl, carboxyl,            C₁₋₆alkoxy, C₁₋₆hydroxyalkoxy, —OC(O)C₁₋₆alkyl,            —N(H)C(O)C₁₋₆alkyl, —N(C₁₋₃alkyl)C(O)C₁₋₆alkyl, amino,            C₁₋₆alkylamino, di(C₁₋₆alkyl)amino, oxo, and azido; or R′            and R″ on the same nitrogen together form a C₃₋₅heterocyclic            ring.

In some embodiments, B¹ and B² are independently selected from:

wherein

Z^(a) is NR′; wherein

-   -   R^(z) is, independently for each occurrence, hydrogen, halogen,        azido, amino, C₁₋₆alkylamino, di(C₁₋₆alkyl)amino, C₁₋₆alkyl,        C₁₋₆alkoxy, —C(O)C₁₋₆alkyl, —CO₂H, —CO₂C₁₋₆alkyl, —C(O)NR′,        —C(O)NR′R″, —OC(O)C₁₋₆alkyl, —NR′C(O)C₁₋₆alkyl, —N(R′)C(O)NR′R″,        or —OC(O)NR′R″, wherein        -   C₁₋₆alkyl and C₁₋₆alkoxy are, independently for each            occurrence, optionally substituted with one or more groups            selected from halogen, thiol, hydroxyl, carboxyl,            C₁₋₆alkoxy, C₁₋₆hydroxyalkoxy, —OC(O)C₁₋₆alkyl,            —N(H)C(O)C₁₋₆alkyl, —N(C₁₋₃alkyl)C(O)C₁₋₆alkyl, amino,            C₁₋₆alkylamino, di(C₁₋₆alkyl)amino, oxo, and azido; and        -   R′ and R″ are, independently for each occurrence, hydrogen            or C₁₋₆alkyl optionally substituted with one or more groups            selected from halogen, thiol, hydroxyl, carboxyl,            C₁₋₆alkoxy, C₁₋₆hydroxyalkoxy, —OC(O)C₁₋₆alkyl,            —N(H)C(O)C₁₋₆alkyl, —N(C₁₋₃alkyl)C(O)C₁₋₆alkyl, amino,            C₁₋₆alkylamino, di(C₁₋₆alkyl)amino, oxo, and azido; or R′            and R″ on the same nitrogen together form a C₃₋₅heterocyclic            ring.

In certain embodiments, B¹ and B² are independently selected from:

In certain embodiments, R^(a1), R^(b1), R^(a2), and R^(b2) are allhydrogen. In some embodiments, at least one of R^(a1), R^(b), R^(a2),and R^(b2) is C₁₋₃alkyl, such as methyl. In certain instances, R^(a1)and R^(a2) are C₁₋₃alkyl, such as methyl. In some embodiments, one ofR^(a1), R^(b1), R^(a2), and R^(b2) is C₁₋₃alkyl, such as methyl.

In certain instances, the CDN has the structure of Formula IIa:

wherein R¹, R², R³, R⁴, R⁵, and R⁶; V¹ and V²; BG¹ and BG²; and B¹ andB²are as defined above for Formula II;or a pharmaceutically acceptable salt thereof.

In some embodiments, of Formula IIa, R¹ is C₂₋₄alkyl, such as ethyl,substituted with hydroxyl, thiol, or amino. In certain embodiments, ofFormula IIa, R¹ is C₂₋₄alkyl, such as ethyl, substituted with thiol oramino.

In certain instances, the CDN has the structure of Formula IIb:

wherein R¹, R⁵, and R⁶; V¹ and V²; BG¹ and BG²; and B¹ and B² are asdefined above for Formula II;or a pharmaceutically acceptable salt thereof.

In some embodiments, of Formula IIb, R¹ is C₂₋₄alkyl, such as ethyl,substituted with hydroxyl, thiol, or amino. In certain embodiments, ofFormula IIb, R¹ is C₂₋₄alkyl, such as ethyl, substituted with thiol oramino.

In certain instances, the CDN has the structure of Formula IIc:

wherein R¹, R⁵, and R⁶; BG¹ and BG²; and B¹ and B² are as defined abovefor Formula II;or a pharmaceutically acceptable salt thereof.

In some embodiments, of Formula IIc, R¹ is C₂₋₄alkyl, such as ethyl,substituted with hydroxyl, thiol, or amino. In certain embodiments, ofFormula IIc, R¹ is C₂₋₄alkyl, such as ethyl, substituted with thiol oramino.

In certain instances, the CDN has the structure of Formula IId:

wherein R¹, R⁵, R⁶, and R^(p); V¹ and V²; and B¹ and B² are as definedabove for Formula II;or a pharmaceutically acceptable salt thereof.

In some embodiments, of Formula IId, R′ is C₂₋₄alkyl, such as ethyl,substituted with hydroxyl, thiol, or amino. In certain embodiments, ofFormula IId, R¹ is C₂₋₄alkyl, such as ethyl, substituted with thiol oramino.

In certain instances, the CDN has the structure of Formula IIe:

wherein R¹, R⁵, R⁶, and R^(p); and B¹ and B² are as defined above forFormula II;or a pharmaceutically acceptable salt thereof.

In some embodiments, of Formula IIe, R¹ is C₂₋₄alkyl, such as ethyl,substituted with hydroxyl, thiol, or amino. In certain embodiments, ofFormula IIe, R¹ is C₂₋₄alkyl, such as ethyl, substituted with thiol oramino.

In certain instances, the CDN has the structure of Formula IIf:

wherein

Y and Z are independently CH or N; and

R¹, R⁵, R⁶, R^(p), and B¹ are as defined above for Formula II;

or a pharmaceutically acceptable salt thereof.

In some embodiments, of Formula IIf, R¹ is C₂₋₄alkyl, such as ethyl,substituted with hydroxyl, thiol, or amino. In certain embodiments, ofFormula IIf, R¹ is C₂₋₄alkyl, such as ethyl, substituted with thiol oramino.

In certain instances, the CDN has the structure of Formula IIg:

wherein

W and X are independently CH or N; and

R¹, R⁵, R⁶, R^(p), and B² are as defined above for Formula II;

or a pharmaceutically acceptable salt thereof.

In some embodiments, of Formula IIg, R¹ is C₂₋₄alkyl, such as ethyl,substituted with hydroxyl, thiol, or amino. In certain embodiments, ofFormula IIg, R¹ is C₂₋₄alkyl, such as ethyl, substituted with thiol oramino.

In certain instances, the CDN has the structure of Formula IIh:

wherein

W, X, Y, and Z are independently CH or N; and

R¹, R⁵, R⁶, and R′ are as defined above for Formula II;

or a pharmaceutically acceptable salt thereof.

In some embodiments, of Formula IIh, R¹ is C₂₋₄alkyl, such as ethyl,substituted with hydroxyl, thiol, or amino. In certain embodiments, ofFormula IIh, R¹ is C₂₋₄alkyl, such as ethyl, substituted with thiol oramino.

In certain instances, the CDN has the structure of Formula IIi:

wherein

W, X, Y, and Z are independently CH or N; and

R¹, R⁵, R⁶, and R^(p) are as defined above for Formula II;

or a pharmaceutically acceptable salt thereof.

In some embodiments, of Formula IIi, R¹ is C₂₋₄alkyl, such as ethyl,substituted with hydroxyl, thiol, or amino. In certain embodiments, ofFormula IIi, R¹ is C₂₋₄alkyl, such as ethyl, substituted with thiol oramino.

In certain instances, the CDN has the structure of Formula IIj:

wherein

W, X, Y, and Z are independently CH or N; and

R¹, R⁵, R⁶, and R^(p) are as defined above for Formula II;

or a pharmaceutically acceptable salt thereof.

In some embodiments, of Formula IIj, R¹ is C₂₋₄alkyl, such as ethyl,substituted with hydroxyl, thiol, or amino. In certain embodiments, ofFormula IIj, R¹ is C₂₋₄alkyl, such as ethyl, substituted with thiol oramino.

In certain instances, the CDN has the structure of Formula IIk:

wherein

W, X, Y, and Z are independently CH or N; and

R¹ and R^(p) are as defined above for Formula II;

or a pharmaceutically acceptable salt thereof.

In some embodiments, of Formula IIk, R¹ is C₂₋₄alkyl, such as ethyl,substituted with hydroxyl, thiol, or amino. In certain embodiments, ofFormula IIk, R¹ is C₂₋₄alkyl, such as ethyl, substituted with thiol oramino.

In certain instances, the CDN has the structure of Formula IIm:

wherein

R¹ is C₂₋₄alkyl, such as ethyl, substituted with a thiol or amino group;

X and Z are independently CH or N;

R^(p), independently for each occurrence, is hydroxyl or thiol;

or a pharmaceutically acceptable salt thereof.

In some embodiments, the CDN has the structure of Formula IIn:

wherein

X and Z are independently CH or N; and

R^(p), independently for each occurrence, is hydroxyl or thiol;

or a pharmaceutically acceptable salt thereof.

In some embodiments, the CDN has the structure of Formula IIo:

wherein

X and Z are independently CH or N; and

R^(p), independently for each occurrence, is hydroxyl or thiol;

or a pharmaceutically acceptable salt thereof.

In some embodiments, in Formulas IId-k and IIm-o, both occurrences ofR^(p) are hydroxyl. In certain embodiments, in Formulas IId-k and IIm-o,both occurrences of R^(p) are thiol. In some embodiments, in FormulasIId-k and IIm-o, the occurrence of R^(p) that corresponds with BG¹ (theupper left R^(p)) is hydroxyl and the occurrence of R^(p) thatcorresponds with BG² (the lower right R^(p)) is thiol. In certainembodiments, in Formulas IId-k and IIm-o, the occurrence of R^(p) thatcorresponds with BG¹ is thiol and the occurrence of R^(p)thatcorresponds with BG² is hydroxyl.

In one embodiment, the CDN has the following structure (CDN-A):

or a pharmaceutically acceptable salt thereof.

In one embodiment, the CDN has the following structure:

or a pharmaceutically acceptable salt thereof.

In one embodiment, the CDN has the following structure:

or a pharmaceutically acceptable salt thereof.

In one embodiment, the CDN has the following structure

or a pharmaceutically acceptable salt thereof.

In one embodiment, the CDN has the following structure (CDN-B):

or a pharmaceutically acceptable salt thereof.

In one embodiment, the CDN has the following structure:

or a pharmaceutically acceptable salt thereof.

In one embodiment, the CDN has the following structure:

or a pharmaceutically acceptable salt thereof.

In one embodiment, the CDN has the following structure

or a pharmaceutically acceptable salt thereof.

The present disclosure provides methods of making ADCs of Formula I byconjugating a CDN of Formula II (e.g., CDN-A or CDN-B) to an antibodyvia a linker. The CDN of Formula II can be conjugated to the antibodyvia a cleavable or non-cleavable linker. In particular embodiments, theCDN is released into a tumor cell, a cancer-related immune cell, or thetumor microenvironment upon cleavage of the linker.

In the ADCs of Formula I, wherein the CDN (D) is of Formula II (e.g.,CDN-A or CDN-B), the CDN may be covalently bound to linker (L) at thehydroxyl, thiol, amino, C₁₋₆alkylamino, or -PEG-OH group of R¹ of theCDN of Formula II. It is understood that the CDN of Formulas II, mayalso not be bound to a linker (L) or ADC and may be be administered inthe methods described herein, either alone, in combination with the ADCsdescribed herein, in combination with other active agents (such asimmuno-oncology agents, such as immune checkpoint inhibitors, includinganti-PD1, anti-PD-L1, and anti-CTLA-4 antibodies), or in combinationboth with the ADCs described herein and in combination with other activeagents (such as immuno-oncology agents).

The CDNs of Formula II (e.g., CDN-A or CDN-B), are capable of agonizingSTING when used alone or as a component of an ADC of Formula I. Inparticular embodiments, the CDNs may be conjugated to an antibody orantigen-binding fragment via a linker. As disclosed herein, the CDNs canbe covalently bonded to a linker via a chemical reaction between thehydroxyl, amino, thiol, C₁₋₆alkylamino, or -PEG-OH group of R¹ of theCDN of Formula II and a corresponding group in the linker. Putdifferently, in some embodiments, the CDN is connected to the linker (L)by one of the following linkages occurring at R¹ of the CDN: R¹—O-L,R¹—NH-L, R¹—S-L, R¹—N(C₁₋₆alkyl)-L, or R¹-PEG-O-L, where R¹ representsthe remainder of the R¹ moiety excluding the hydroxyl, amino, thiol,C₁₋₆alkylamino, or -PEG-OH group of R¹. Particular antibodies,antigen-binding fragments, and linkers are described below.

6.3. Antibodies and Antigen-Binding Fragments

As used herein, the term “antibody” refers to an immunoglobulin moleculethat specifically binds to a particular target antigen. Antibodies maybe of human or non-human origin. Antibodies may be conjugated to CDNsdescribed in Section 6.2 via a linker. The antibodies may be polyclonal,monoclonal, genetically engineered, and/or otherwise modified in nature.The antibodies composing the ADCs of the disclosure are suitable foradministration to humans, for example, as humanized antibodies or fullyhuman antibodies.

Antibodies comprise heavy and light chains having hypervariable regionsknown as complementarity determining regions (CDRs) that mediate bindingof the antibody with the target antigen. Antibodies generally comprise aheavy chain comprising a variable region (V_(H)) having three CDRs,namely, V_(H) CDR #1, V_(H) CDR #2, and V_(H) CDR #3, and a light chaincomprising a variable region (V_(L)) having three CDRs, namely, V_(L)CDR #1, V_(L) CDR #2, and V_(L) CDR #3. Specific embodiments of the ADCsof the disclosure include, but are not limited to, those that compriseantibodies and/or antigen-binding fragments that include these exemplaryCDRs and/or V_(H) and/or V_(L) sequences.

Antibodies composing ADCs of the disclosure may be in the form offull-length antibodies that may be of, or derived from any antibodyisotype, including for example, IgA, IgD, IgE, IgG, IgM, or IgY. In someembodiments, the antibody composing the ADCs is an IgG (e.g., IgG₁,IgG₂, IgG₃, or IgG₄). In some embodiments, the antibodies comprise allor a portion of a constant region of an antibody.

Antibodies composing ADCs of the disclosure may be bispecificantibodies, dual variable domain antibodies, multiple chain or singlechain antibodies, single domain antibodies, camelized antibodies,scFv-Fc antibodies, surrobodies (including surrogate light chainconstruct) and the like.

The ADCs of the disclosure may comprise full-length (intact) antibodymolecules, as well as antigen-binding fragments. As used herein, theterm “fragment” refers to a portion of an intact antibody that comprisesfewer amino acid residues than the intact antibody. As used herein, theterm “antigen-binding fragment” refers to a polypeptide fragment of anantibody that mediates binding to an antigen, or competes with intactantibody for antigen-binding. Suitable exemplary antigen-bindingfragments include Fab, Fab′, F(ab′)2, Fv, scFv, dAb, Fd, or an isolatedcomplementarity determining region (CDR) having sufficient framework tobind. As would be appreciated by a skilled artisan, fragments can beobtained by molecular engineering or via chemical or enzymatic treatmentof an intact antibody or antibody chain or by recombinant means.

Antibodies or antigen-binding fragments thereof are not limited to aparticular method of generation or production, and can be prepared usingwell known techniques such as hybridoma techniques, recombinanttechniques, phage display technologies, transgenic animals, or somecombination thereof.

6.4. Target Antigens and Antibodies

The antibodies or antigen-binding fragments thereof composing the ADCsas contemplated in the present disclosure specifically bind to one ormore cancer related tumor or immune cell associated antigens.

In certain embodiments, the cancer related tumor or immune cellassociated antigen is a T-cell co-inhibitory molecule. In someembodiments, the antibody or antigen-binding fragment thereofspecifically binds to a tumor associated antigen selected from PD-L1,PD-L2, CD47, CD80, CD86, HVEM, UL144, CD155, CD112, CD113, galectin-1,galectin-3, galectin-9, CD48, LIGHT, BTLA, and CD160. In someembodiments, the tumor associated antigen is a molecule that binds to aT-cell molecule selected from BTLA, Tim-3, PD-1, CTLA-4, TIGIT, CD244,and CD223.

In some embodiments, the antibody is an anti-PD-L1 antibody, such asatezolizumab, durvalumab, avelumab, or antigen-binding fragment thereofor an antibody or antigen-binding fragment thereof having an aminosequence equivalent thereto.

In some embodiments, the antibody is an anti-CD47 antibody, such asHu5F9-G4, IBI188, CC-90002, ZL1201, TTI-621, AO-176, the antibody ofSGN-CD47M, the antigen binding domain of ALX148, or antigen-bindingfragment thereof or an antibody or antigen-binding fragment thereofhaving an amino acid sequence equivalent thereto.

In other embodiments, the antibody or antigen-binding fragment thereofspecifically binds to a cancer related tumor antigen which is a GrowthFactor Receptor (GFR). In certain embodiments, the cancer related tumorantigen is an EGFR/ErbB/HER family GFR. In some embodiments, the cancerrelated tumor antigen is selected from an EGFR/HER1 (ErbB1), HER2/c-Neu(ErbB2), Her3 (ErbB3), and Her4 (ErbB4) receptor. In certainembodiments, the cancer related tumor antigen is an IGFR family GFR. Insome embodiments, the cancer related tumor antigen is an IGF1R or IGF2Rreceptor. In certain embodiments, the cancer related tumor antigen is aTGF-βR (TβR) family GFR. In some embodiments, the cancer related tumorantigen is a TβR I or TβR II receptor. In certain embodiments, thecancer related tumor antigen is a VEGFR family GFR. In some embodiments,the cancer related tumor antigen is a VEGFR1, VEGFR2, or VEGFR3receptor. In certain embodiments, the cancer related tumor antigen is aPDGFR family GFR. In some embodiments, the cancer related tumor antigenis a PDGFR-α, or PDGFR-β receptor. In certain embodiments, the cancerrelated tumor antigen is a FGFR family GFR. In some embodiments, thecancer related tumor antigen is a FGFR1, FGFR2, FGFR3, or FGFR4receptor.

In some embodiments, the antibody is an anti-EGFR/HER1 (ErbB1) antibody,such as cetuximab, panitumumab, necitumumab, or antigen-binding fragmentthereof or an antibody or antigen-binding fragment thereof having anamino sequence equivalent thereto. In some embodiments, the antibody isan anti-HER2 (ErbB2) antibody, such as trastuzumab, pertuzumab, orantigen-binding fragment thereof or an antibody or antigen-bindingfragment thereof having an amino sequence equivalent thereto. In someembodiments, the antibody is an anti-VEGFR2 antibody, such asramucirumab, or antigen-binding fragment thereof or an antibody orantigen-binding fragment thereof having an amino sequence equivalentthereto. In some embodiments, the antibody is an anti-PDGFR-α antibody,such as olaratumab, or antigen-binding fragment thereof or an antibodyor antigen-binding fragment thereof having an amino sequence equivalentthereto.

In other embodiments, the antibody or antigen-binding fragment thereofspecifically binds to lymphoma related antigen. In certain embodiments,the lymphoma related antigen is CD20, CD30, CD19/CD3, CD22, or CD33.

In some embodiments, the antibody is an anti-CD20 antibody, such asrituximab, ibritumomab, ofatumumab, obinutuzumab, or antigen-bindingfragment thereof or an antibody or antigen-binding fragment thereofhaving an amino sequence equivalent thereto. In some embodiments, theantibody is an anti-CD30 antibody, such as brentuximab orantigen-binding fragment thereof or an antibody or antigen-bindingfragment thereof having an amino sequence equivalent thereto. In someembodiments, the antibody is an anti-CD19/CD3 antibody, such asblinatumomab, or antigen-binding fragment thereof or an antibody orantigen-binding fragment thereof having an amino sequence equivalentthereto. In some embodiments, the antibody is an anti-CD22 antibody,such as inotuzumab, or antigen-binding fragment thereof or an antibodyor antigen-binding fragment thereof having an amino sequence equivalentthereto. In some embodiments, the antibody is an anti-CD33 antibody,such as gemtuzumab, or antigen-binding fragment thereof or an antibodyor antigen-binding fragment thereof having an amino sequence equivalentthereto.

In other embodiments, the antibody or antigen-binding fragment thereofspecifically binds to myeloma related antigen. In certain embodiments,the lymphoma related antigen is SLAMF7 or CD38.

In some embodiments, the antibody is an anti-SLAMF7 antibody, such aselotuzumab or antigen-binding fragment thereof or an antibody orantigen-binding fragment thereof having an amino sequence equivalentthereto. In some embodiments, the antibody is an anti-CD38 antibody,such as daratumumab or antigen-binding fragment thereof or an antibodyor antigen-binding fragment thereof having an amino sequence equivalentthereto.

In other embodiments, the antibody or antigen-binding fragment thereofspecifically binds to blastoma related antigen. In certain embodiments,the blastoma related antigen is GD2.

In some embodiments, the antibody is an anti-GD2 antibody, such asdinutuximab, or antigen-binding fragment thereof or an antibody orantigen-binding fragment thereof having an amino sequence equivalentthereto.

In other embodiments, the antibody or antigen-binding fragment thereofspecifically binds to RANK Ligand.

In some embodiments, the antibody is an anti-RANK Ligand antibody, suchas denosumab, or antigen-binding fragment thereof or an antibody orantigen-binding fragment thereof having an amino sequence equivalentthereto.

In certain embodiments, the antibody is an antibody that binds to anantigen preferentially expressed or overexpressed in cancer cells, suchas PD-L1 and EGFR.

In some embodiments, the antibody is an antibody that binds to anantigen derived from a microbe that infects human cells.

As used herein, “a” and “anti” are used interchangeably, for example aswhen describing an “anti-PD-L1” antibody or “α-PD-L1” antibody.

As used herein, protein names having hyphenation are usedinterchangeably with their non-hyphenated form (i.e., “PD-L1” and “PDL1”are used interchangeably).

As used herein, numbering of immunoglobulin amino acid residues is doneaccording to the Eu numbering system, unless otherwise indicated.

6.5. Linkers

In the ADCs described herein, the CDN is linked to the antibody orantigen-binding fragment by way of a multi-atom linker. The linkers linkthe CDN to the antibody or the antigen-binding fragment by forming acovalent linkage to CDN at one location on the linker and a covalentlinkage to the antibody or antigen-binding fragment at another locationon the linker. The linkers may be monovalent with respect to the CDN(e.g., in Formula Ta), such that they covalently link a single CDN to asingle site on the antibody or fragment thereof. The linkers may also bepolyvalent with respect to the CDN (e.g., in Formula I when m>1), suchthat they covalently link more than one CDN to a single site on theantibody or fragment thereof. As used herein, the expression “linker” isintended to include unconjugated, partially conjugated (i.e., to the CDNor Ab only), and fully conjugated forms of the linker (i.e., to both theCDN and Ab). In specific embodiments, moieties comprising functionalgroups on the antibody and linker which form the covalent linkagebetween the antibody and the linker are specifically illustrated asR^(X) and R^(Y), respectively.

The linkers linking the CDN to the antibody or fragment thereof may belong, short, flexible, rigid, hydrophilic, or hydrophobic in nature, ormay comprise segments that have different characteristics. A widevariety of linkers useful for linking drugs to antibodies or fragmentsthereof in the context of ADCs are known in the art. These linkers, aswell as other linkers, may be used to link the CDN to the antibody ofantigen-binding fragment of the ADCs described herein.

In certain embodiments, linkers include from 2-100, 2-75, 2-50, 2-25,2-10, 5-100, 5-75, 5-50, 5-25, 5-10, 10-100, 10-75, 10-50, or 10-25atoms in the chain that connects the CDN to the antibody orantigen-binding fragment (including any atoms at the ends of the linkerthat may derive from the CDN or antibody or antigen-binding fragment).Likewise, linkers used in CDN-coupled linkers (as discussed below), alsomay include from 2-100, 2-75, 2-50, 2-25, 2-10, 5-100, 5-75, 5-50, 5-25,5-10, 10-100, 10-75, 10-50, or 10-25 atoms in the chain that connectsthe CDN to a site on the linker capable of coupling to a complementarysite on an antibody or antigen-binding fragment.

The linker may be chemically stable to extracellular environment andserum, or may include linkages that are intentionally unstable and canrelease the CDN in the extracellular milieu or tumor microenvironment.

In some embodiments, the linkers include linkages that are designed torelease the CDN upon internalization of the ADC within a cell. In somespecific embodiments, the linkers include linkages designed to cleaveand/or immolate or otherwise specifically or non-specifically degradeinside cells.

The number of CDNs linked to the antibody or antigen-binding fragmentthereof of an ADC can vary (called the “drug-to-antibody ratio,” or“DAR”) and will be limited by the number of available attachments siteson the antibody or antigen-binding fragment thereof and the number ofCDNs linked to a single linker. In ADCs that include more than one CDN,each CDN may be the same or different. As long as the CDN does notexhibit unacceptable levels of aggregation under the conditions of useand/or storage, ADCs with DARs of 10, or even higher, are contemplated.In some embodiments, the ADCs described herein may have a DAR in therange of 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, or 1 to 4. Insome embodiments, the ADCs described herein may have a DAR in the rangeof 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, or 2 to 4. Incertain specific embodiments, the ADCs may have a DAR of 1, 2, 3, or 4.In other specific embodiments, the ADCs may have a DAR of 5, 6, 7, or 8.In some specific embodiments, the ADCs may have a DAR of 1.

By way of example and not limitation, some cleavable and non-cleavablelinkers that may be included in the ADCs described herein are describedbelow.

6.5.1. Cleavable Linkers

In certain embodiments, the linker is cleavable in vivo by chemical orenzymatic processes to liberate the CDN. In certain instances, the CDNis cleaved from the linker to regenerate the same CDN prior to couplingwith the linker. In other embodiments, the liberated CDN is a CDNmodified from the CDN that was originally coupled to the linker, themodified CDN having a residual functional group from the linker butretaining efficacy or even exhibiting enhanced efficacy over theoriginal CDN. Generally, cleavable linkers incorporate one or morechemical bonds that are either chemically or enzymatically cleavable,while the remainder of the linker is non-cleavable.

In certain embodiments, the cleavable linker comprises a chemicallylabile group. Chemically labile groups exploit differential propertiesbetween the plasma and some cytoplasmic compartments, for example theacidic environment of endosomes and lysosomes, or the high thiolconcentrations in the cytosol (e.g., glutathione). In certainembodiments, the plasma stability of a linker comprising a chemicallylabile group may be increased or decreased by altering steric hindrancenear the chemically labile group using substituents.

In some embodiments, the chemically labile group of the cleavable linkeris an acid labile group. Acid labile groups can remain intact duringcirculation in the blood's neutral pH and undergo hydrolysis underacidic conditions to release the CDN, such as within an acidic tumormicroenvironment or upon internalization into endosomal (pH 5.0-6.5) andlysosomal (pH 4.5-5.0) cellular compartments. This pH-dependent releasemechanism of the cleavable linker can be optimized by chemicalmodification, e.g., substitution, to tune release of CDN to a particularpH. In some embodiments, the cleavable linker comprises an acid-labilegroup, such as a hydrazone, hydrazine, cis-aconityl, acetal, orthoester,or an imine group. In some embodiments, the acid-labile group undergoescleavage in the tumor microenvironment, in an endosome of the tumor orimmune cell, in a lysosome of the tumor or immune cell, in an acidicintracellular compartment of the tumor or immune cell, or anycombination thereof. In some embodiments, the acid-labile group does notundergo cleavage in the tumor microenvironment, in an endosome of thetumor or immune cell, in a lysosome of the tumor or immune cell, and/orin an acidic intracellular compartment of the tumor or immune cell. Ascontemplated herein, cleavability of the acid-labile group may bedetermined by pH sensitivity of the fully conjugated linker in the ADC.Acid labile linkers may contain additional cleavage sites, such asadditional acid-labile cleavage sites and/or enzymatically labilecleavage sites.

In certain embodiments, the cleavable linker comprises a disulfidegroup. Disulfides are designed to release the drug upon internalizationinto cells, where the cytosol provides a more reducing environmentcompared to the extracellular environment. Scission of disulfide bondsgenerally requires the presence of a cytosolic thiol cofactor, such as(reduced) glutathione (GSH), such that disulfide-containing linkers arereasonably stable in circulation and selectively release the drug in thecytosol. The intracellular enzyme protein disulfide isomerase, orsimilar enzymes capable of cleaving disulfide bonds, may also contributeto disulfide cleavage. Tumor cells may induce a hypoxic state due toirregular blood flow, resulting in enhanced reductive enzyme activityand even higher glutathione concentrations.

ADCs including exemplary disulfide-containing linkers include thefollowing formulas:

wherein

D represents the CDN (e.g., CDN-A or CDN-B);

S of S-D is from the CDN, NH of Ab-NH is from the Ab, S of Ab-S is fromthe Ab;

Ab represents the antibody or binding fragment thereof;

“n” represents the number of number of occurrences of D linked to Ab viathe linker; and

R is, independently for each occurrence, hydrogen or C₁₋₃alkyl.

In certain embodiments, the linker comprises the structure

where the NH of *—NH may be from the Ab, and

represents the point of attachment of the linker directly or indirectlyto the CDN (e.g., at the hydroxyl, amino, thiol, etc. of R¹ of the CDN,e.g., CDN-A or CDN-B). In certain embodiments,

represents a direct point of attachment of the linker to a thiol of theR¹ group of the CDN, wherein the S adjacent

is part of the thiol of the R¹ group.

Another type of cleavable linker contemplated for the disclosed ADCs isan enzymatically cleavable linker. Such linkers are typicallypeptide-based or include peptidic regions, and can be more stable inplasma and extracellular milieu than chemically labile linkers. Peptidebonds are generally stable in serum due to a higher pH value compared tolysosomes and the presence of endogenous inhibitors of lysosomalproteolytic enzymes.

Release of a CDN from the ADC can occur by action of lysosomalproteases, e.g., cathepsin and plasmin, which may be present at elevatedlevels in certain tumor cells. In some embodiments, the cleavablepeptide is cleaved by a lysosomal enzyme. In certain embodiments, thecleavable peptide is cleaved by a cathepsin (e.g. Cathepsin B) orplasmin.

As a skilled artisan would recognize, proteolytic cleavage of a peptidelinker that is directly attached to a CDN can produce an amino acidadduct of the CDN upon amide bond hydrolysis. Thus, also contemplatedfor ADCs of the disclosure is an enzymatically cleavable linker thatcomprises a self-immolative spacer to spatially separate the CDN fromthe cleavage site. The use of a self-immolative spacer allows for theelimination of the fully active, chemically unmodified CDN of Formula IIupon amide bond hydrolysis.

One contemplated self-immolative spacer is the bifunctionalpara-aminobenzyl alcohol group, which on one end is linked at thebenzylic hydroxyl group to an amine group on the CDN functionalized witha carbamate, and on the other end is linked at the amino group to forman amide bond with the peptide (i.e., a PABC group). Uponprotease-mediated cleavage of the peptide, the resulting CDN isactivated, leading to a 1,6-elimination reaction that releases theunmodified CDN, carbon dioxide, and remnants of the linker. In someembodiments, the cleavable linker comprises a PABC group. Additionallycontemplated self-immolative spacers are heterocyclic variants of PABCthat have been described (see for example, U.S. Pat. No. 7,989,434,which is incorporated herein by reference).

In some embodiments, the enzymatically cleavable linker is anon-peptidic linker. In certain embodiments, the non-peptidic linker ispeptidomimetic. In certain embodiments, the non-peptidic linker iscleaved by tumor-specific proteases. In certain embodiments, thenon-peptidic linker is cleaved by tumor-specific proteases havingincreased abundance in tumors and/or the tumor microenvironment). Incertain embodiments, the non-peptidic linker is cleaved by cathepsin B.In certain embodiments, the non-peptidic linker iscyclobutane-1,1-dicarboxamide.

In some embodiments, the enzymatically cleavable linker is aβ-glucuronic acid-based linker. Cleavage of the β-glucuronide glycosidicbond can occurs via the lysosomal enzyme β-glucuronidase, which isabundantly present within lysosomes and is overexpressed in some tumortypes, while having low activity outside cells.

Cleavable linkers may include non-cleavable portions or segments, and/orcleavable segments or portions may be included in an otherwisenon-cleavable linker to render it cleavable. By way of example only,polyethylene glycol (PEG) and related polymers may include cleavablegroups in the polymer backbone. For example, a polyethylene glycol orpolymer linker may include one or more cleavable groups such as adisulfide, a hydrazine, a hydrazone, a dipeptide, or acyclobutane-1,1-dicarboxamide.

In certain embodiments, the linker comprises an enzymatically cleavablepeptide moiety, for example, a linker comprising Formula VIIIa, VIIIb,VIIIc, VIIId, VIIIe, VIIIf, or VIIIg:

wherein:

“peptide” represents a peptide or peptidomimetic chain (illustrated N→Cand not showing the carboxyl and amino “termini”) cleavable by alysosomal enzyme;

T represents a chain (e.g., a polymer chain) comprising one or moreethylene glycol units or an alkylene chain, or combinations thereof;

R^(a) is selected from hydrogen, C₁₋₆alkyl, sulfonate, and methylsulfonate;

R^(b) is selected from hydrogen,

n is an integer ranging from 2 to 10, such as 3 to 6, particularly 5;

p is an integer ranging from 0 to 5;

q is an integer ranging from 0 to 5, particularly 3;

w is 0 or 1, and the —S— of *—S— may be from the Ab;

x is 0 or 1, and the NH of *—NH may be from the Ab;

y is 0 or 1; and the NH of *—NH may be from the Ab;

z is 0 or 1;

represents the point of attachment of the linker directly or indirectlyto the CDN (e.g., at the hydroxyl, amino, thiol, etc. of R¹ of the CDN,e.g., CDN-A or CDN-B); and

* represents the point of attachment to the remainder of the linker ordirectly or indirectly to the antibody;

or a salt thereof.

In some embodiments, the cleavable peptide moiety or “peptide” inFormulas VIIIa-d comprise the following structure:

where the terminal —NH— may be from the Ab if x, y, and w are 0.

In some embodiments, the cleavable peptide moiety or “peptide” inFormula VIIIe comprises the following structure:

where w is 1.

In certain embodiments, the cleavable peptide moiety or “peptide” inFormulas VIIIa-g comprises from 2-20 amino acid residues, such as 2-15,2-10, 2-7, or 2-5 residues, including a tetrapeptide, a tripeptide, or adipeptide. In particular embodiments, the cleavable peptide moiety or“peptide” comprises a dipeptide, such as a dipeptide selected from:Ala-Ala, Ala-(D)Asp, Ala-Cit, Ala-Lys, Ala-Val, Asn-Cit, Asp-Cit,Asn-Lys, Asn-(D)Lys, Asp-Val, Cit-Ala, Cit-Asn, Cit-Asp, Cit-Cit,Cit-Lys, Cit-Ser, Cit-Val, Glu-Val, PhenylGly-(D)Lys, His-Val, Ile-Cit,Ile-Pro, Ile-Val, Leu-Cit, Lys-Cit, Me3Lys-Pro, Met-Lys, Met-(D)Lys,Phe-Arg, Phe-Cit, Phe-Lys, Pro-(D)Lys, Ser-Cit, Trp-Cit, Val-Ala,Val-(D)Asp, NorVal-(D)Asp, Val-Cit, Val-Glu, Val-Lys, and salts thereof.In certain embodiments, the dipeptide is Val-Cit. In certainembodiments, the cleavable peptide moiety or “peptide” comprises atripeptide, such as Glu-Val-Cit. In certain embodiments, the cleavablepeptide moiety or “peptide” comprises a tetrapeptide, such asGly-Phe-Leu-Gly or Ala-Leu-Ala-Leu.

In certain embodiments, the linker is of Formula VIIIa and comprises thefollowing structure:

where “peptide” is Glu-Val-Cit.

In certain embodiments, the linker is of Formula VIIIc and comprises thefollowing structure:

where “peptide” is Val-Cit.

In certain embodiments, the cleavable peptide moiety or “peptide” inFormula VIIId comprises dipeptide, such as a dipeptide selected from:Ala-Ala, Ala-(D)Asp, Ala-Cit, Ala-Lys, Ala-Val, Asn-Cit, Asp-Cit,Asn-Lys, Asn-(D)Lys, Asp-Val, Cit-Ala, Cit-Asn, Cit-Asp, Cit-Cit,Cit-Lys, Cit-Ser, Cit-Val, Glu-Val, PhenylGly-(D)Lys, His-Val, Ile-Cit,Ile-Pro, Ile-Val, Leu-Cit, Lys-Cit, Me3Lys-Pro, Met-Lys, Met-(D)Lys,Phe-Arg, Phe-Cit, Phe-Lys, Pro-(D)Lys, Ser-Cit, Trp-Cit, Val-Ala,Val-(D)Asp, NorVal-(D)Asp, Val-Cit, Val-Glu, Val-Lys, and salts thereof.In certain embodiments, the dipeptide in Formula VIIId is Val-Cit. Incertain embodiments, the cleavable peptide moiety or “peptide” inFormula VIIId comprises a tripeptide, such as Glu-Val-Cit. In certainembodiments, the cleavable peptide moiety or “peptide” in Formula VIIIdcomprises a tetrapeptide, such as Gly-Phe-Leu-Gly or Ala-Leu-Ala-Leu.

In certain embodiments, the linker is of Formula VIIId and comprises thefollowing structure:

where “peptide” is Val-Cit.

In certain embodiments, the linker is of Formula VIIIe and comprises thefollowing structure:

where “peptide” is

6.5.2. Groups Used to Attach Linkers to Antibodies

A variety of attachment groups may be used to attach linker-CDN synthonsto antibodies to produce ADCs. Attachment groups on the linker-CDNsynthon are generally electrophilic in nature. In some embodiments, theattachment group is selected from a maleimide group; an activateddisulfide such as DSDM, SPDB, or sulfo-SPDB; an active ester such as anNHS ester or a HOBt ester; a haloformate, an acid halide, and an alkylor benzyl halide such as a haloacetamide. In certain embodiments, theresulting linkage between the linker (L) and the antibody (Ab) is athioether, an amide, an ester, a carbamate, a carbonate, a urea, adisulfide, or an ether.

Also contemplated for the disclosed ADCs are “self-stabilizing”maleimides and “bridging disulfides”. An example of a “self-stabilizing”maleimide group is provided for in US 2013/0309256, hereby incorporatedby reference. Examples of “bridging disulfides” are provided for inBadescu et al., 2014, Bioconjugate Chem. 25:1124-1136, and WO2013/085925, each of which are hereby incorporated by reference. 6.5.3.ADCs with Cathepsin Cleavable Linkers

As set forth above, in some embodiments of the disclosure, the CDN(e.g., CDN-A or CDN-B) and the antibodies comprising the ADCs of thedisclosure are linked via a cathepsin cleavable linker. In one suchembodiment, the ADC has the structure of Formula III:

wherein variables W, X, Y, Z, R^(p), and n are defined as above forFormulas I and II. In the schematic above,

represents covalent linkage of the cathepsin cleavable linker to theantibody or antigen-binding fragment thereof (Ab).

In one embodiment, the pyrrolidine-2,5-dione group of the linker ofFormula III is linked at its 3-position to the antibody (Ab) by a thiolgroup. For instance, the pyrrolidine-2,5-dione can be covalently linkedat its 3-position to the antibody via a cysteine residue on theantibody. The resultant ADC has the structure of Formula IIIa:

wherein variables W, X, Y, Z, R^(p), and n are defined as above forFormulas I and II.

In one embodiment, the ADC has the following structure:

6.5.4. ADCs with Glutathione Cleavable Linkers

As set forth above, in some embodiments of the disclosure, the CDN(e.g., CDN-A or CDN-B) and the antibodies comprising the ADCs of thedisclosure are linked via a glutathione cleavable linker. In one suchembodiment, the ADC has the structure of Formula IV:

wherein variables W, X, Y, Z, R^(p), and n are defined as above inFormulas I and II. In the schematic above,

represents covalent linkage of the glutathione cleavable linker to theantibody (Ab).

In one embodiment, the carbonyl group of the linker in Formula IV can becovalently linked to the antibody via a lysine or other amino acidresidue on the antibody bearing an amino group-containing sidechain,such as by forming an amide bond with the amino group at the

bond attached to the carbonyl group. The resultant ADC has the structureof Formula IVa:

wherein variables W, X, Y, Z, R^(p), and n are defined as above inFormulas I and II.

In one embodiment, the ADC has the following structure:

6.6. Methods of Making Antibody-Drug Conjugates

Generally, ADCs according to Formula I may be prepared according to thefollowing scheme:Ab-R^(X)+R^(Y)-L-(D)_(m)→(Formula I) Ab-[-L-(D)_(m)]_(n)wherein Ab, L, D, m, and n are as previously defined for Formula I, andR^(X) and R^(Y) represent complementary groups capable of formingcovalent linkages with one another.

Relatedly, ADCs according to Formula Ta may be prepared according to thefollowing scheme:Ab-R^(X)+R^(Y)-L-D→(Formula Ia) Ab-[-L-D]_(n)wherein Ab, L, D, and n are as previously defined for Formula I, andR^(X) and R^(Y) represent complementary groups capable of formingcovalent linkages with one another, as described above.

The identities of groups R^(X) and R^(Y) will depend upon the chemistryused to link synthon R^(Y)-L-(D)_(m) or R^(Y)-L-D to the antibody. Thesynthons are typically linked to the side chains of amino acid residuesof the antibody, including, for example, a free thiol group of anaccessible cysteine residue or a primary amino group of an accessiblelysine residue. In some embodiments, R^(X) is a group on a side chain ofan amino acid of the antibody, such as an amino or thiol group. Incertain embodiments, R^(X) is an amino group on a side chain of an aminoacid, such as lysine, 5-hydroxylysine, ornithine, or statine,particularly lysine. In some embodiments, R^(X) is a thiol group on aside chain of an amino acid, such as cysteine or homocysteine,particularly cysteine. In such linkages, free thiol groups may beobtained by first fully or partially reducing the antibody to disruptinterchain disulfide bridges between cysteine residues. A number offunctional groups R^(Y) and chemistries may be used to form linkageswith thiol groups, and include by way of example and not limitationmaleimides and haloacetyls.

Also contemplated for the disclosed ADCs are engineered antibodieshaving mutations to or more codons to disrupt one or more disulfidebridges, and includes by way of example and not limitation the mutationof a single cysteine residue of an interchain disulfide bridge to aserine residue to produce a free thiol from the unpaired cysteine. Alsocontemplated for the disclosed ADCs are engineered antibodies havingmutations to or more codons to introduce a residue having a thiol forlinker conjugation, and includes by way of example and not limitationthe mutation of one or more residues to a cysteine residue, orincorporation of additional cysteine residues into the amino acidsequence of the antibody or antigen-binding fragment thereof.

In certain embodiments, R^(X) is a thiol, such as from a cysteineresidue on the antibody, and R^(Y) is a group selected from ahaloacetyl, maleimide, aziridine, acryloyl, vinylsulfone, pyridyldisulfide, TNB-thiol, and an alkylating or arylating agent. In someembodiments, R^(X) is a thiol group, such as from a cysteine residue onthe antibody, and R^(Y) is a maleimide group. In some embodiments, R^(X)is a primary amino group of a lysine residue. A number of functionalgroups R^(X) and chemistries may be used for lysine linkages, andinclude by way of example and not limitation NHS-esters andisothiocyanates.

In certain embodiments, R^(X) is an amine, such as from a lysine residueon the antibody, and R^(Y) is a group capable of alkylating or acylatingthe amine. In some embodiments, R^(X) is an amine, such as from a lysineresidue on the antibody, and R^(Y) is a group selected from anisothiocyanate, isocyanate, acyl azide, NHS ester, sulfonyl chloride,aldehyde, glyoxal, epoxide, oxirane, carbonate, aryl halide, imidoestes,carbodiimide, anhydride, and a fluorophenyl ester. In certainembodiments, R^(X) is an amine, such as from a lysine residue on theantibody, and R^(Y) is an NHS ester.

As contemplated for ADCs of the disclosure, conjugation chemistries arenot limited to available side chain groups. An antibody may also beengineered to include amino acid residues for conjugation, and includesby way of example and not limitation the conversion of side chains suchas amines to other useful groups, such as a thiol, by linking anappropriate small molecule to the amine. For instance, a primaryamine-containing side chain of an amino acid may be converted to athiol-containing side chain, such as —NH—C₁₋₆alkyl-SH, including—NH—CH₂—CH₂—SH, where —NH— is from the primary amine.

As will be appreciated by skilled artisans, the number of CDNs linked toan antibody molecule may vary, resulting in a heterogeneous ADCpreparation in which some antibodies contain one linked CDN, some two,some three, etc. (and some none). The degree of heterogeneity willdepend upon, among other things, the chemistry used for linking the CDN.For example, when an IgG1 antibody is reduced to yield thiol groups forattachment, heterogeneous mixtures of antibodies having zero, 1, 2, 3,4, 5, 6, 7, or 8 linked CDNs per molecule are often produced.Furthermore, by adjusting the molar ratio of R^(X) to R^(Y), ADCs having0, 1, 2, 3, 4, 5, 6, 7, or 8 linked CDNs per molecule can be produced.Thus, it will be understood that depending upon context, stateddrug-to-antibody ratios (DARs) may be averages for a collection of ADCs.For example, “DAR3” refers to a heterogeneous ADC preparation in whichthe average drug-to-antibody ratio is 3, e.g., a mixture of ADCs havingequal numbers of DAR2 and DAR4.

Purity may be assessed by a variety of methods, as is known in the art.As a specific example, an ADC preparation may be analyzed via HPLC,hydrophobic exchange, ion exchange, size exclusion, or otherchromatography and the purity assessed by analyzing areas under thecurves of the resultant peaks.

In some embodiments, the present disclosure is directed to a method ofmaking an ADC comprising (a) coupling one or more CDNs of the disclosure(i.e., a CDN of Formula II) to a linker (e.g., L as described herein) togenerate one or more CDN-coupled linkers; and (b) coupling one or moreof the CDN-coupled linkers to an antibody or antigen-binding fragmentthereof (e.g., Ab as described herein) to generate the ADC.

In certain embodiments, the present disclosure is directed to a methodof making an ADC comprising (b) coupling one or more of CDN-coupledlinkers (i.e., a CDN of Formula II and, e.g., L as described herein) toan antibody or antigen-binding fragment thereof (e.g., Ab as describedherein) to generate the ADC.

In certain embodiments, the present disclosure is directed to a methodof making one or more CDN-coupled linkers comprising (a) coupling one ormore CDNs of the disclosure (i.e., a CDN of Formula II) to a linker(e.g., L as described herein) to generate one or more CDN-coupledlinkers.

In other embodiments, the present disclosure is directed to a method ofmaking an ADC comprising (a) coupling one or more linkers (e.g., L asdescribed herein) to an antibody or antigen-binding fragment thereof(e.g., Ab as described herein) to generate a linker-coupled antibody;and (b) coupling one or more CDNs of the disclosure (i.e., a CDN ofFormula II) to the linker-coupled antibody to generate the ADC.

In certain embodiments, the present disclosure is directed to a methodof making an ADC comprising (b) coupling one or more CDNs of thedisclosure (i.e., a CDN of Formula II) to a linker-coupled antibody orantigen-binding fragment thereof (e.g., one or more L as describedherein and, e.g., Ab as described herein) to generate the ADC.

In certain embodiments, the present disclosure is directed to a methodof making a linker-coupled antibody comprising (a) coupling one or morelinkers (e.g., L as described herein) to an antibody or antigen-bindingfragment thereof (e.g., Ab as described herein) to generate thelinker-coupled antibody.

In some embodiments, the present disclosure is directed to a method ofmaking an ADC comprising (a) coupling a CDN of the disclosure (i.e., aCDN of Formula II) to a linker (e.g., L as described herein) to generatea CDN-coupled linker; and (b) coupling a plurality of the CDN-coupledlinkers to an antibody or antigen-binding fragment thereof (e.g., Ab asdescribed herein) to generate the ADC.

In other embodiments, the present disclosure is directed to a method ofmaking an ADC comprising (a) coupling a plurality of linkers (e.g., L asdescribed herein) to an antibody or antigen-binding fragment thereof(e.g., Ab as described herein) to generate a linker-coupled antibody;and (b) coupling a plurality of one or more CDNs of the disclosure(i.e., a CDN of Formula II) to the linker-coupled antibody to generatethe ADC.

6.6.1. CDN Compositions for Handling CDNs to Prior to Coupling

As described above, CDNs of the disclosure (i.e., a CDN of Formula II,such as CDN-A or CDN-B) may be coupled to a linker (e.g., L as describedherein) to generate one or more CDN-coupled linkers, or may be coupledto a linker-coupled antibody (e.g., one or more L as described hereinand, e.g., Ab as described herein) to generate an ADC. Therefore, thepresent disclosure provides CDN compositions that facilitate thepurification, drying, or handling of the CDNs that are used in thesecoupling steps.

In that context, the present disclosure provides a CDN compositioncomprising a CDN of Formula II (including sub formulas of Formula II,such as Formula IIa, IIb, etc.), and a base, such as an amine base. Insome embodiments, the amine base is a liquid at room temperature andpressure, such as pyridine, piperidine, pyrrolidine, morpholine,lutidine (e.g., 2,6-lutidine), triethylamine (TEA), ordiisopropylethylamine (DIPEA), particularly pyridine.

In certain embodiments, the CDN composition comprising the CDN and thebase is an anhydrous composition having less than 100, 50, 25, or 10 ppmof water. The CDN composition comprising the CDN and the base can bedried by concentration under high vacuum prior to use in a couplingstep.

In some embodiments, the CDN composition comprises a CDN of Formula II,such as Formula IIk, IIm, IIn, or IIo, and an amine base, such aspyridine. In certain embodiments, the CDN composition comprises a CDN ofFormula II that has an amino group at the R¹ position, and an aminebase, such as pyridine. In certain embodiments, the CDN compositioncomprises a CDN of Formula IIn or IIo (and optionally that has an aminogroup at the R¹ position), and an amine base, such as pyridine. In someembodiments, the CDN composition comprises CDN-A as described herein, ora pharmaceutically acceptable salt thereof, and an amine base, such aspyridine, and is optionally anhydrous. In some embodiments, the CDNcomposition comprises CDN-B as described herein, or a pharmaceuticallyacceptable salt thereof, and an amine base, such as pyridine, and isoptionally anhydrous.

The present disclosure also provides a CDN composition that is anaqueous solution comprising a CDN of Formula II (including sub formulasof Formula II, such as Formula IIa, IIb, etc.), and a buffer suitable toachieve a pH in the range of 5 to 10, including 5 to 8, such as a pH of5, 5.5, 5.8, 6, 6.2, 6.5, 7, 7.5 or 8, including any pH ranges createdby using these pH values as end points, such as 5 to 7. In certainembodiments, the buffer is suitable to achieve a pH of 6+/−0.2. In someembodiments, the buffer is a phosphate buffer. In some embodiments, theCDN composition is an aqueous solution comprising a CDN of Formula IIthat has a thiol group at the R¹ position, such as Formula IIo, and abuffer suitable to achieve a pH of 6+/−0.2, such as a phosphate buffer.In particular embodiments, the CDN composition is an aqueous solutioncomprising a CDN of Formula IIo and a buffer suitable to achieve a pH of6+/−0.2, such as a phosphate buffer. In one embodiment, the CDNcomposition is an aqueous solution comprising CDN-B or apharmaceutically acceptable salt thereof and a buffer suitable toachieve a pH of 6+/−0.2, particularly a phosphate buffer.

6.6.2. CDN Compositions for Coupling CDNs to Linkers

Preparation of a CDN-coupled linker, as described above (i.e.,comprising a CDN of Formula II, such as CDN-A or CDN-B, coupled to alinker, e.g., L as described herein), may include preparation of one ormore CDN compositions for coupling the CDN to the linker.

In that context, the present disclosure provides a CDN compositioncomprising a CDN of Formula II (including sub formulas of Formula II,such as Formula IIa, IIb, etc.) and either a linker (e.g., L asdescribed herein) or a coupling agent, or both a linker and a couplingagent, wherein the coupling agent facilitates coupling of the CDN to thelinker. In some embodiments, the coupling agent is capable of activatingthe linker for coupling with the CDN, such as by generating an activatedester on the linker such that the CDN is then capable of reacting withthe activated ester of the linker to couple the CDN to the linker.Examples of suitable coupling agents include hydroxybenzotriazole(HOBt), N-hydroxysuccinimide (NHS), dicyclohexylcarbodiimide (DCC),diisopropylcarbodiimide (DIC),1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), andhexafluorophosphate azabenzotriazole tetramethyl uranium (HATU). Incertain embodiments, the CDN composition further comprises an aproticpolar solvent, such as dimethylformamide (DMF), dimethylacetamide (DMA),acetonitrile, or tetrahydrofuran (THF), particularly DMF. In certainembodiments, the CDN composition comprising the CDN and either or bothof the linker and the coupling agent is an anhydrous composition havingless than 100, 50, 25, or 10 ppm of water.

In certain embodiments, the CDN composition comprises a CDN of FormulaII, such as Formula IIk, IIm, IIn, or IIo (and has an amino or thiolgroup at the R¹ position), and either a linker (e.g., L as describedherein) or a coupling agent or both a linker and a coupling agent. Insome embodiments, the CDN composition comprises a CDN of Formula II thathas an amino or thiol group at the R¹ position, and either a linker or acoupling agent or both a linker and a coupling agent. In certainembodiments, the CDN composition comprises a CDN of Formula IIn or IIo(and optionally that has an amino or thiol group at the R¹ position),and either a linker or a coupling agent or both a linker and a couplingagent. In certain embodiments, the CDN composition comprises CDN-A orCDN-B as described herein, or a pharmaceutically acceptable salt ofeither, and either a linker or a coupling agent, or both a linker and acoupling agent, and optionally an aprotic polar solvent as describedabove. In certain embodiments, the CDN composition comprises CDN-A orCDN-B or a pharmaceutically acceptable salt of either, a linker, and anaprotic polar solvent. In certain embodiments, the CDN compositioncomprises CDN-A or CDN-B or a pharmaceutically acceptable salt ofeither, a coupling agent, and an aprotic polar solvent.

6.6.3. CDN-Coupled Linkers

As discussed above, CDN-coupled linkers may be useful intermediates inthe preparation of ADCs described herein. In some embodiments, aCDN-coupled linker comprises a CDN of Formula II (e.g., CDN-A or CDN-B)coupled to a linker, e.g., L as described herein. For example, aCDN-coupled linker may have the formula L-CDN, wherein L includes a sitecapable of coupling to a complementary site on an antibody orantigen-binding fragment, wherein the CDN is of Formula II (includingsub formulas, such as Formulas Ilk, IIm, IIn, and IIo), and wherein theCDN is covalently bound to the linker at the hydroxyl, thiol, amino,C₁₋₆alkylamino, or -PEG-OH group of the R¹ position of Formula II.

In certain embodiments, the CDN and the linker of a CDN-coupled linkerare coupled via a thioether, an amide, an ester, a carbamate, acarbonate, a urea, a disulfide, or an ether group, particularly anamide, carbamate, or disulfide group. For example, in certainembodiments, the CDN and the linker of a CDN-coupled linker are coupledvia an amide group. In such embodiments, the CDN-coupled linker may havethe formula L-C(O)NH-CDN or L-C(O)N(C₁₋₆alkyl)-CDN, wherein the CDN isof Formula II (including sub formulas, such as Formulas Ilk, IIm, andIIn), wherein R¹ of Formula II is C₁₋₆alkyl, such as C₂₋₆alkyl orC₂₋₃alkyl, substituted with an amino or a C₁₋₆alkylamino group, eitherof which form the amine portion of the amide group in the formula. Inone such embodiment, the CDN-coupled linker has the formulaL-C(O)NH-CDN, wherein the CDN is of Formula II (including sub formulas,such as Formulas Ilk, IIm, and IIn), wherein R¹ of Formula II isC₂₋₃alkyl, such as ethyl, substituted with an amino group, which formsthe amine portion of the amide group in the formula.

In some embodiments, the CDN-coupled linker has the structure of FormulaIXa:

wherein R^(L) represents the remainder of the linker L, and variables W,X, Y, Z, and R^(p) are defined as above for Formulas I and II.

In other embodiments, the CDN and the linker of a CDN-coupled linker arecoupled via a carbamate group. In such embodiments, the CDN-coupledlinker may have the formula L-OC(O)NH-CDN or L-OC(O)N(C₁₋₆alkyl)-CDN,wherein the CDN is of Formula II (including sub formulas, such asFormulas Ilk, IIm, and IIn), wherein R¹ of Formula II is C₁₋₆alkyl, suchas C₂₋₆alkyl or C₂₋₃alkyl, substituted with an amino or a C₁₋₆alkylaminogroup, either of which form the amine portion of the carbamate group inthe formula. In one such embodiment, the CDN-coupled linker has theformula L-OC(O)NH-CDN, wherein the CDN is of Formula II (including subformulas, such as Formulas Ilk, IIm, and IIn), wherein R¹ of Formula IIis C₂₋₃alkyl, such as ethyl, substituted with an amino group, whichforms the amine portion of the carbamate group in the formula.

In some embodiments, the CDN-coupled linker has the structure of FormulaIXb:

wherein R^(L) represents the remainder of the linker L, and variables W,X, Y, Z, and R^(p) are defined as above for Formulas I and II.

In some embodiments, the CDN-coupled linker has the following structure:

wherein variables W, X, Y, Z, and R^(p) are defined as above forFormulas I and II.

In other embodiments, the CDN and the linker of a CDN-coupled linker arecoupled via a urea group. In such embodiments, the CDN-coupled linkermay have the formula L-NHC(O)NH-CDN or L-NHC(O)N(C₁₋₆alkyl)-CDN, whereinthe CDN is of Formula II (including sub formulas, such as Formulas Ilk,IIm, and IIn), wherein R¹ of Formula II is C₁₋₆alkyl, such as C₂₋₆alkylor C₂₋₃alkyl, substituted with an amino or a C₁₋₆alkylamino group,either of which form the rightmost amine portion of the urea group inthe formula. In one such embodiment, the CDN-coupled linker has theformula L-NHC(O)NH-CDN, wherein the CDN is of Formula II (including subformulas, such as Formulas Ilk, IIm, and IIn), wherein R¹ of Formula IIis C₂₋₃alkyl, such as ethyl, substituted with an amino group, whichforms the rightmost amine portion of the urea group in the formula.

In some embodiments, the CDN-coupled linker has the structure of FormulaIXc:

wherein R^(L) represents the remainder of the linker L, and variables W,X, Y, Z, and R^(p) are defined as above for Formulas I and II.

In other embodiments, the CDN and the linker of a CDN-coupled linker arecoupled via an ester group. In such embodiments, the CDN-coupled linkermay have the formula L-C(O)O-CDN, wherein the CDN is of Formula II(including sub formulas, such as Formulas Ilk, IIm, and IIn), wherein R¹of Formula II is C₁₋₆alkyl, such as C₂₋₆alkyl or C₂₋₃alkyl, substitutedwith a hydroxyl or a -PEG-OH group, either of which form the alcoholportion of the ester group in the formula. In one such embodiment, theCDN-coupled linker has the formula L-C(O)O-CDN, wherein the CDN is ofFormula II (including sub formulas, such as Formulas Ilk, IIm, and IIn),wherein R¹ of Formula II is C₂₋₃alkyl, such as ethyl, substituted with ahydroxyl group, which forms the alcohol portion of the ester group inthe formula.

In some embodiments, the CDN-coupled linker has the structure of FormulaIXd:

wherein R^(L) represents the remainder of the linker L, and variables W,X, Y, Z, and R^(p) are defined as above for Formulas I and II.

In other embodiments, the CDN and the linker of a CDN-coupled linker arecoupled via a carbonate group. In such embodiments, the CDN-coupledlinker may have the formula L-OC(O)O-CDN, wherein the CDN is of FormulaII (including sub formulas, such as Formulas Ilk, IIm, and IIn), whereinR¹ of Formula II is C₁₋₆alkyl, such as C₂₋₆alkyl or C₂₋₃alkyl,substituted with a hydroxyl or a -PEG-OH group, either of which form therightmost alcohol portion of the carbonate group in the formula. In onesuch embodiment, the CDN-coupled linker has the formula L-OC(O)O-CDN,wherein the CDN is of Formula II (including sub formulas, such asFormulas Ilk, IIm, and IIn), wherein R¹ of Formula II is C₂₋₃alkyl, suchas ethyl, substituted with a hydroxyl group, which forms the rightmostalcohol portion of the carbonate group in the formula.

In some embodiments, the CDN-coupled linker has the structure of FormulaIXe:

wherein R^(L) represents the remainder of the linker L, and variables W,X, Y, Z, and R^(p) are defined as above for Formulas I and II.

In other embodiments, the CDN and the linker of a CDN-coupled linker arecoupled via a disulfide group. In such embodiments, the CDN-coupledlinker may have the formula L-S—S-CDN, wherein the CDN is of Formula II(including sub formulas, such as Formulas Ilk, IIm, and IIo), wherein R¹of Formula II is C₁₋₆alkyl, such as C₂₋₆alkyl or C₂₋₃alkyl, substitutedwith a thiol group, which forms the rightmost portion of the disulfidegroup in the formula. In one such embodiment, the CDN-coupled linker hasthe formula L-S—S-CDN, wherein the CDN is of Formula II (including subformulas, such as Formulas Ilk, IIm, and IIo), wherein R¹ of Formula IIis C₂₋₃alkyl, such as ethyl, substituted with a thiol group, which formsthe rightmost portion of the disulfide group in the formula.

In some embodiments, the CDN-coupled linker has the structure of FormulaIXf:

wherein R^(L) represents the remainder of the linker L, and variables W,X, Y, Z, and R^(p) are defined as above for Formulas I and II.

In some embodiments, the CDN-coupled linker has the following structure:

wherein variables W, X, Y, Z, and R^(p) are defined as above forFormulas I and II.

6.7. Pharmaceutical Compositions and Medicaments

The ADCs and/or or CDNs described herein may be in the form ofpharmaceutical compositions comprising the ADC or CDN and one or morecarriers, excipients and/or diluents. The compositions may be formulatedfor pharmaceutical use in humans, and may include a pharmaceuticallyacceptable carrier, such as an aqueous, optionally buffered, solution,suspension, or dispersion. The compositions may also be lyophilizedsolid compositions, which may be reconstituted prior to use.

The present pharmaceutical compositions can be in any suitable form, andcan be administered to a patient by a variety of routes such asintravenously, intratumorally, subcutaneously, intramuscularly, orally,intranasally, intrathecally, transdermally, topically, or locally. Theroute for administration in any given case may depend on the particularantibody and/or ADC, the subject, and the nature and severity of thedisease and the physical condition of the subject. In certainembodiments, the pharmaceutical composition will be administeredintravenously, intratumorally, subcutaneously, or intramuscularly in theform of a liquid formulation.

In some embodiments, the ADCs and/or CDN's described herein, includingtheir pharmaceutical compositions, are administered systemically, suchas subcutaneously, intraperitoneally, intramuscularly, or intravenously,particularly intravenously. In other embodiments, the ADCs and/or CDN'sdescribed herein, including their pharmaceutical compositions, areadministered locally at a tumor site, such as intratumorally or in themicroenvironment of the tumor.

The present disclosure also provides CDNs of Formula II (including subformulas of Formula II, such as Formula IIa, IIb, etc.) for use in themanufacture of medicaments for therapy, such as for promoting an immuneresponse and/or for treating cancer in a subject, including one or moreof the various cancers described below, and in combination with one ormore additional therapeutic agents as described below.

6.8. Methods of Treatment

In certain embodiments, an ADC of the disclosure (i.e., an ADC ofFormula I) or a CDN of the disclosure (i.e., a CDN of Formula II, suchas CDN-A or CDN-B) may be used in therapy. In some embodiments, thepresent disclosure is directed to pharmaceutical compositions describedherein comprising an ADC of the disclosure (i.e., an ADC of Formula I)or a CDN of the disclosure (i.e., a CDN of Formula II) for use intherapy.

The ADCs and/or CDNs disclosed herein may be employed alone or incombination with each other and/or with other therapeutic agents. Asshown in the examples discussed below, the ADCs and CDNs are capable ofpromoting an immune response when delivered to a subject. For instance,the ADCs and CDNs of the disclosure, either alone or in combination arecapable of inducing interferon-β (IFNβ) in a human subject. The abilityof the ADCs and CDNs of the disclosure to promote an immune response isattributed, in part, to their ability to agonize STING. The ADCs arecapable of delivering CDNs of the disclosure (e.g., a compound ofFormula II) to target tumor or cancer-related immune cells, or the tumormicroenvironment to trigger activation of STING and the resultant immuneresponse. Conjugation of the CDNs to an antibody or antigen-bindingfragment thereof that binds to a cancer-related tumor or immune cellantigen targets delivery of the CDN and prolongs and enhances the immuneresponse.

The ADCs of the disclosure are capable of promoting an immune responsethat is greater than either the unconjugated CDN or the antibody thatcomprises the ADC. Surprisingly, it has been found that the ADCs of thedisclosure can promote an immune response that is greater than theadditive immune response of the unconjugated CDN and the antibodycomprising the ADC. In other words, by conjugating a CDN of thedisclosure with a specific immunotherapeutic antibody, a synergisticeffect can be achieved. In other embodiments, as discussed herein, theCDNs of the disclosure may also be administered not as part of an ADC.

Accordingly, in one aspect, the disclosure provides methods of inducingor promoting an immune response in a subject comprising administering aneffective amount of an ADC of the disclosure (i.e., an ADC of FormulaI). In another aspect, the disclosure provides methods of inducing orpromoting an immune response in a subject comprising administering aneffective amount of a CDN of the disclosure (i.e., a CDN of Formula II).And in another aspect, the disclosure provides methods of inducing orpromoting an immune response in a subject comprising administering aneffective amount of an ADC of the disclosure in combination with a CDNof the disclosure. In some embodiments, the ADC and/or CDN areadministered to mammals in need thereof. In particular embodiments, theADC and/or CDN are administered to humans in need thereof.

In particular embodiments, the ADCs or CDNs of the present disclosureare used to treat cancer. For instance, the ADCs or CDNs of thedisclosure can be used to treat cancers of the lung, bone, pancreas,skin, head, neck, uterus, ovaries, stomach, colon, breast, esophagus,small intestine, bowel, endocrine system, thyroid gland, parathyroidgland, adrenal gland, urethra, prostate, penis, testes, ureter, bladder,kidney or liver. Further cancers treatable by the ADCs or CDNs of thepresent disclosure include rectal cancer; cancer of the anal region;carcinomas of the fallopian tubes, endometrium, cervix, vagina, vulva,renal pelvis, and renal cell; sarcoma of soft tissue; myxoma;rhabdomyoma; fibroma; lipoma; teratoma; cholangiocarcinoma;hepatoblastoma; angiosarcoma; hemagioma; hepatoma; fibrosarcoma;chondrosarcoma; myeloma; chronic or acute leukemia; lymphocyticlymphomas; primary CNS lymphoma; neoplasms of the CNS; spinal axistumours; squamous cell carcinomas; synovial sarcoma; malignant pleuralmesotheliomas; brain stem glioma; pituitary adenoma; bronchial adenoma;chondromatous hanlartoma; inesothelioma; Hodgkin's Disease; or acombination of one or more of the foregoing cancers.

Accordingly, in one aspect, the disclosure provides methods of treatingcancer in a subject comprising administering a pharmaceuticalcomposition comprising a pharmaceutically acceptable amount of an ADC ofthe disclosure (i.e., an ADC of Formula I). In another aspect, thedisclosure provides methods of treating cancer in a subject comprisingadministering a pharmaceutical composition comprising a pharmaceuticallyacceptable amount of a CDN of the disclosure (i.e., a CDN of FormulaII). In some embodiments, the pharmaceutical compositions areadministered to mammals in need thereof. In particular embodiments, thepharmaceutical compositions are administered to humans in need thereof.

In another aspect, the disclosure provides methods of treating cancer ina subject by administering a pharmaceutical composition comprising apharmaceutically acceptable amount of an ADC of the disclosure (i.e., anADC of Formula I) with at least one additional anti-cancer agent to asubject (e.g., a human). The ADC and the one or more additionalanti-cancer agents may be administered together or separately and, whenadministered separately, administration may occur simultaneously orsequentially, in any order, by any convenient route in separate orcombined pharmaceutical compositions. In some embodiments, theadditional anti-cancer agent enhances expression of the target antigenof the antibody or antigen-binding fragment thereof of the ADC ofFormula I. The amounts of the ADC and the other pharmaceutically activeanti-cancer agent(s) and the relative timings of administration will beselected in order to achieve the desired combined therapeutic effect.

In some instances, the disclosure provides methods of treating cancer ina subject by administering a pharmaceutical composition comprising apharmaceutically acceptable amount of a CDN of the disclosure (i.e., aCDN of Formula II) with at least one additional anti-cancer agent to asubject (e.g., a human). The CDN and the one or more additionalanti-cancer agents may be administered together or separately and, whenadministered separately, administration may occur simultaneously orsequentially, in any order, by any convenient route in separate orcombined pharmaceutical compositions. The amounts of the CDN and theother anti-cancer agent(s) and the relative timings of administrationwill be selected in order to achieve the desired combined therapeuticeffect.

The combination of an ADC or CDN of the disclosure and one or moreanti-cancer agents may be administered together in a singlepharmaceutical composition. Alternatively, the ADC or CDN and the one ormore anti-cancer agents may be formulated separately. When formulatedseparately they may be provided in any convenient composition,conveniently, in such a manner as known for such compounds in the art.

Accordingly, an ADC or CDN of the disclosure may be employed with othertherapeutic methods of cancer treatment, e.g., in anti-neoplastictherapy, combination therapy with immune checkpoint inhibitors, otherchemotherapeutic, hormonal, antibody agents as well as surgical and/orradiation treatments, particularly radiation.

In one embodiment, an ADC of the disclosure (i.e., an ADC of Formula I)or a CDN of the disclosure (i.e., a CDN of Formula II) is—or both an ADCand a CDN of the disclosure are—employed in combination with an immunecheckpoint inhibitor to treat cancer. Immune checkpoint inhibitors, suchas humanized antibodies against PD-1, PD-L1, and CTLA4, have recentlybeen shown to be highly successful in treating several types ofmetastatic cancer, including melanoma, non-small cell lung cancers,renal cell carcinoma and bladder cancer (Sharma and Allison, 2015,Science 348, 56). However, still only a small percentage of cancerpatients benefit from the checkpoint inhibitor therapies, in partbecause insufficient number of anti-tumor immune cells, such as CD8 Tcells, are generated and/or infiltrated into the tumors. As shown inexamples described herein, the combination of an ADC and/or CDN of thedisclosure and an immune checkpoint inhibitor is capable of functioningsynergistically to treat cancers that are refractory to monotherapy withthe immune checkpoint inhibitor.

In one embodiment, the disclosure provides methods of treating cancer ina subject by administering a pharmaceutical composition comprising apharmaceutically acceptable amount of an ADC of the disclosure (i.e., anADC of Formula I) or a CDN of the disclosure (i.e., a CDN of Formula II)or both an ADC and CDN of the disclosure in combination with a PD-L1inhibitor. Examples of PD-L1 inhibitors that can be used in combinationwith ADCs of the disclosure include, but are not limited to,atezolizumab (Tecentriq®), avelumab (Bavencio®), durvalumab (Imfinzi®),BMS-936559, and CK-301. In certain embodiments, the ADC and/or CDN ofthe disclosure is not administered in combination with a PD-L1inhibitor, including those mentioned above.

In one embodiment, the disclosure provides methods of treating cancer ina subject by administering a pharmaceutical composition comprising apharmaceutically acceptable amount of an ADC of the disclosure (i.e., anADC of Formula I) or a CDN of the disclosure (i.e., a CDN of Formula II)or both an ADC and CDN of the disclosure in combination with a PD-1inhibitor. Examples of PD-1 inhibitors that can be used in combinationwith ADCs of the disclosure include, but are not limited to,pembrolizumab (Keytruda®), nivolumab (Opdivo®), cemiplimab (Libtayo®),AMP-224, AMP-514, and PDR001. In certain embodiments, the ADC and/or CDNof the disclosure is not administered in combination with a PD-1inhibitor, including those mentioned above.

In one embodiment, the disclosure provides methods of treating cancer ina subject by administering a pharmaceutical composition comprising apharmaceutically acceptable amount of an ADC of the disclosure (i.e., anADC of Formula I) or a CDN of the disclosure (i.e., a CDN of Formula II)or both an ADC and CDN of the disclosure in combination with a CTLA-4inhibitor. Examples of CTLA-4 inhibitors that can be used in combinationwith ADCs of the disclosure include, but are not limited to, ipilmumab(Yervoy®) and tremelimumab. In certain embodiments, the ADC and/or CDNof the disclosure is not administered in combination with a CTLA-4inhibitor, including those mentioned above.

In another embodiment, the disclosure provides methods of treatingcancer in a subject by administering a pharmaceutical compositioncomprising a pharmaceutically acceptable amount of an ADC of thedisclosure (i.e., an ADC of Formula I) or a CDN of the disclosure (i.e.,a CDN of Formula II) or both an ADC and CDN of the disclosure with oneor more anti-microtubule agents such as diterpenoids and vincaalkaloids; platinum coordination complexes; alkylating agents such asnitrogen mustards, oxazaphosphorines, alkylsulfonates, nitrosoureas, andtriazenes; antibiotic agents such as anthracyclins, actinomycins andbleomycins; topoisomerase II inhibitors such as epipodophyllotoxins;antimetabolites such as purine and pyrimidine analogues and anti-folatecompounds; topoisomerase I inhibitors such as camptothecins; hormonesand hormonal analogues; signal transduction pathway inhibitors;non-receptor tyrosine angiogenesis inhibitors; immunotherapeutic agents;proapoptotic agents; and or cell cycle signaling inhibitors.

The ADCs of Formula I can be used, e.g., for treating cancer or forinducing or promoting an immune response, in combination with a STINGagonist that is not conjugated to an antibody or antigen-bindingfragment. In certain embodiments, the STING agonist that is notconjugated to an antibody or antigen-binding fragment is a CDN, such asone of those described herein, i.e., 2′3′-CDNs. In other embodiments,the STING agonist is a 3′3′-CDN, a 2′2′-CDN, or a 3′2′-CDN. In someembodiments, the STING agonist is a benzophenone analog. In furtherembodiments, the STING agonist is a dimeric aminobenzimidazole. Examplesof STING agonists that are not conjugated to an antibody orantigen-binding fragment that can be used in combination with ADCs ofthe disclosure include, IMSA101, ADU-S100 (MIW815), BMS-986301, CRD5500,CMA (10-carboxymethyl-9-acridanone), diABZI STING agonist-1 (e.g., CASNo.: 2138299-34-8), DMXAA (ASA404/vadimezan), E7766, GSK-532,GSK-3745417, MK-1454, MK-2118, SB-11285, SRCB-0074, TAK-676, andTTI-10001. The STING agonist can be administered prior to, concurrentlywith or following administration of the ADC of Formula I.

The ADCs of Formula I can be used, e.g., for treating cancer or forinducing or promoting an immune response, in combination with a “free”CDN that is not conjugated to the antibody or antigen-binding fragmentof Formula I. The free CDN can be administered prior to, concurrentlywith or following administration of the ADC of Formula I. In such cases,the free CDN may be the same or different than the CDN that isconjugated to the antibody of the ADC of Formula I. The free CDN may bea cGAMP, e.g., 2′3′-cGAMP or an analog or derivative thereof or apharmaceutically acceptable salt thereof. In other embodiments, the freeCDN is a 3′3′-cGAMP, 2′2′-cGAMP, 3′2′-cGAMP or an analog or derivativeof any of these or a pharmaceutically acceptable salt thereof.

Accordingly, the disclosure provides methods of treating cancer in asubject by administering a pharmaceutical composition comprising apharmaceutically acceptable amount of an ADC of the disclosure (i.e., anADC of Formula I) with at least one CDN that is not conjugated to anantibody (“free CDN”). The ADCs of Formula I and the free CDN may beadministered together or separately and, when administered separately,administration may occur simultaneously or sequentially, in any order,by any convenient route in separate or combined

In one embodiment, the free CDN administered in combination with the ADCof Formula I is the following compound, or a pharmaceutically acceptablesalt thereof:

In another embodiment, the free CDN administered in combination with theADC of Formula I is the following compound, or a pharmaceuticallyacceptable salt thereof:

In another embodiment, the free CDN administered in combination with theADC of Formula I is the following compound, or a pharmaceuticallyacceptable salt thereof:

In another embodiment, the free CDN administered in combination with theADC of Formula I is the following compound, or a pharmaceuticallyacceptable salt thereof:

In another embodiment, the free CDN administered in combination with theADC of Formula I is the following compound, or a pharmaceuticallyacceptable salt thereof:

In another embodiment, the free CDN administered in combination with theADC of Formula I is the following compound, or a pharmaceuticallyacceptable salt thereof:

In another embodiment, the free CDN administered in combination with theADC of Formula I is the following compound, or a pharmaceuticallyacceptable salt thereof:

In another embodiment, the free CDN administered in combination with theADC of Formula I is the following compound, or a pharmaceuticallyacceptable salt thereof:

In another embodiment, the free CDN administered in combination with theADC of Formula I is the following compound, or a pharmaceuticallyacceptable salt thereof:

In another embodiment, the free CDN administered in combination with theADC of Formula I is the following compound, or a pharmaceuticallyacceptable salt thereof:

In another embodiment, the free CDN administered in combination with theADC of Formula I is the following compound, or a pharmaceuticallyacceptable salt thereof:

In another embodiment, the free CDN administered in combination with theADC of Formula I is the following compound, or a pharmaceuticallyacceptable salt thereof:

In another embodiment, the free CDN administered in combination with theADC of Formula I is the following compound, or a pharmaceuticallyacceptable salt thereof:

In another embodiment, the free CDN administered in combination with theADC of Formula I is the following compound, or a pharmaceuticallyacceptable salt thereof:

In another embodiment, the free CDN administered in combination with theADC of Formula I is the following compound, or a pharmaceuticallyacceptable salt thereof:

In another embodiment, the free CDN administered in combination with theADC of Formula I is the following compound, or a pharmaceuticallyacceptable salt thereof:

In another embodiment, the free CDN administered in combination with theADC of Formula I is the following compound, or a pharmaceuticallyacceptable salt thereof:

In another embodiment, the free CDN administered in combination with theADC of Formula I is the following compound, or a pharmaceuticallyacceptable salt thereof:

In another embodiment, the free CDN administered in combination with theADC of Formula I is the following compound, or a pharmaceuticallyacceptable salt thereof:

7. EXAMPLES

The following Examples, which highlight certain features and propertiesof exemplary embodiments of CDNs, ADCs, and methods of using these ADCsto treat patients are provided for purposes of illustration, and notlimitation.

Abbreviations

¹H-NMR Proton nuclear magnetic resonance spectroscopy

¹⁹F-NMR ¹⁹F nuclear magnetic resonance spectroscopy

³¹P-NMR ³¹P nuclear magnetic resonance spectroscopy

G Guanine

A Adenine

A^(Bz) 6N-benzoyladenine

Gib 2N-isobutyryl

Bz Benzoyl

DCA Dichloroacetic acid

DCM Dichloromethane

DMOCP 2-chloro-5,5-dimethyl-1,3,2-dioxaphosphineane 2-oxide

DMT 4,4′-dimethoxytrityl

DMTCl 4,4′-dimethoxytrityl chloride

PBS Phosphate-buffered saline

Py. Pyridine

TBS t-Butyldimethylsilyl

TrCl Tris(hydroxymethyl)aminomethane hydrochloride

IBX 2-Iodoxybenzoic acid

LAH Lithium aluminum hydride

DMF Dimethylformamide

NMM N-Methylmorpholine

Et₃N Triethylamine Example 1. Preparation of CDN-A

Schemes A1 and A2 below depict the synthesis of a CDN (“CDN-A”)disclosed herein. The synthesis and characterization of the CDN and thesynthetic intermediates are described below.

Synthesis of Intermediate 19 from 1

Step 1: Synthesis of(3aR,5R,6S,6aR)-5-(hydroxymethyl)-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-6-ol

A solution of (2R,4S,5R)-5-(hydroxymethyl)tetrahydrofuran-2,3,4-triol(100 g, 0.67 mol) in acetone (2.6 L) containing H₂SO₄ (con., 184.00 g,1.88 mol, 100 mL, 2.8 eq.) is stirred at 20° C. for 1 h. A solution ofNa₂CO₃ (130 g, 1.23 mol, 1.8 eq.) in H₂O (600 mL) is carefully added at0° C. The mixture is stirred for a further 2.5 h before a second batchof Na₂CO₃ (70 g, 0.66 mol) is added. After 0.5 h, the precipitate iscollected by filtration and washed by acetone (0.5 L×3). The filtrate isconcentrated and purified by column chromatography (SiO₂, DCM:MeOH=10:1to 5:1) to give(3aR,5R,6R)-5-(hydroxymethyl)-2,2-dimethyl-3a,5,6,6a-tetrahydrofuro[2,3-d][1,3]dioxol-6-ol(53.5 g, 0.28 mol, 85% yield) as yellow oil. (MS: [M+Na]⁺ 213.0).

Step 2: Synthesis of3aR,5R,6S,6aR)-2,2-dimethyl-5-((trityloxy)methyl)tetrahydrofuro[2,3-d][1,3]dioxol-6-ol

To a solution of(3aR,5R,6R)-5-(hydroxymethyl)-2,2-dimethyl-3a,5,6,6a-tetrahydrofuro[2,3-d][1,3]dioxol-6-ol(125 g, 0.66 mol) in pyridine (600 mL) is added TrCl (219.9 g, 0.79 mol,1.2 eq.). After 16 h at 60° C., the mixture is cooled down andconcentrated. The residue is partitioned between CH₂Cl₂ (400 mL) and aq.NaHCO₃(sat., 800 mL). The aqueous phase is extracted with CH₂Cl₂ (600mL×2). The combined organic layers are dried over Na₂SO₄, filtered,concentrated and purified by column chromatography (SiO₂, petroleumether/ethyl acetate=10:1 to 5:1) to give(3aR,5R,6R)-2,2-dimethyl-5-(trityloxymethyl)-3a,5,6,6a-tetrahydrofuro[2,3-d][1,3]dioxol-6-ol(250 g, 0.58 mol, 88% yield) as white solid. (MS: [M+Na]⁺ 455.0).

Step 3: Synthesis of(3aR,5R,6aS)-2,2-dimethyl-5-((trityloxy)methyl)dihydrofuro[2,3-d][1,3]dioxol-6(3aH)-one

To a solution of(3aR,5R,6R)-2,2-dimethyl-5-(trityloxymethyl)-3a,5,6,6a-tetrahydrofuro[2,3-d][1,3]dioxol-6-ol(250 g, 0.58 mol) in CH₃CN (1.5 L) is added IBX (323 g, 1.2 mol, 2.00eq.). The mixture is stirred at 90° C. for 6 h. After cooling down, themixture is filtered. The filtrate is concentrated and give(3aR,5R,6aS)-2,2-dimethyl-5-(trityloxymethyl)-3a,6a-dihydrofuro[2,3-d][1,3]dioxol-6-one(240 g, 0.56 mol, 96.5% yield) as light yellow oil. (MS: [M+Na]⁺ 453.0).

Step 4: Synthesis of(E)-methyl-2-((3aR,5S,6aR)-2,2-dimethyl-5-((trityloxy)methyl)furo[2,3-d][1,3]dioxol-6(3aH,5H,6aH)-ylidene)acetate

To a mixture of NaH (14.5 g, 0.36 mol, 60% in oil, 1.3 eq.) and THF(1.00 L) is added methyl 2-dimethoxyphosphorylacetate (66 g, 0.36 mol,52.4 mL, 1.3 eq.) dropwise at 0° C. over 15 min. The mixture is stirredat the same temperature for 45 min before a solution of(3aR,5R,6aS)-2,2-dimethyl-5-(trityloxymethyl)-3a,6a-dihydrofuro[2,3-d][1,3]dioxol-6-one(120 g, 0.28 mol, 1 eq.) in THF (500 mL) is added dropwise. After 15 hat 25° C., the reaction is quenched by NH₄Cl (sat., 50 mL) at 0° C. Themixture is concentrated and partitioned between brine (500 mL) and CH₂C₂(500 mL×3). The combined organic layers are dried over Na₂SO₄, filtered,concentrated and purified by column chromatography (SiO₂, petroleumether/ethyl acetate=15:1 to 5:1) to give methyl(2E)-2-[(3aR,5S,6aR)-2,2-dimethyl-5-(trityloxymethyl)-3a,6a-dihydrofuro[2,3-d][1,3]dioxol-6-ylidene]acetate(65 g, 0.53 mol, 96% yield) as light yellow oil. (MS: [M+Na]⁺ 509.0).

Step 5: Synthesis of methyl2-((3aR,5S,6R,6aR)-2,2-dimethyl-5-((trityloxy)methyl)tetrahydrofuro[2,3-d][1,3]dioxol-6-yl)acetate

To a solution of methyl(2E)-2-[(3aR,5S,6aR)-2,2-dimethyl-5-(trityloxymethyl)-3a,6a-dihydrofuro[2,3-d][1,3]dioxol-6-ylidene]acetate(260 g, 0.53 mol) in EtOAc (700 mL) is added Pd/C (10% on carbon, 10 g)under N₂ atmosphere. The suspension is degassed and purged with H₂ for 3times. The mixture is stirred under H₂ (20 psi) at 25° C. for 16 h. Thecatalyst is removed by filtration. The filtrate is concentrated andpurified by column chromatography (SiO₂, petroleum ether/ethylacetate=15:1 to 10:1) to give methyl2-[(3aR,5S,6S)-2,2-dimethyl-5-(trityloxymethyl)-3a,5,6,6a-tetrahydrofuro[2,3-d][1,3]dioxol-6-yl]acetate(210 g, 0.43 mol, 80.4% yield) as white solid. (MS: [M+Na]⁺ 511.1).

Step 6: Synthesis of2-((3aR,5S,6R,6aR)-2,2-dimethyl-5-((trityloxy)methyl)tetrahydrofuro[2,3-d][1,3]dioxol-6-yl)ethanol

To a mixture of LiAlH₄ (15.5 g, 0.41 mol, 2 eq.) and THF (500 mL) isslowly added a solution of methyl2-[(3aR,5S,6S)-2,2-dimethyl-5-(trityloxymethyl)-3a,5,6,6a-tetrahydrofuro[2,3-d][1,3]dioxol-6-yl]acetate(100 g, 0.20 mol) in THF (20 mL) at 0° C. After being stirred for 2.5 hat 25° C., the reaction is quenched by water (15 mL) and NaOH (aq., 15%,15 mL) at 0° C. The crude is dried over Na₂SO₄, filtered, concentratedand purified by column chromatography (SiO₂, petroleum ether/ethylacetate=5:1 to 2:1) to give2-[(3aR,5S,6S)-2,2-dimethyl-5-(trityloxymethyl)-3a,5,6,6a-tetrahydrofuro[2,3-d][1,3]dioxol-6-yl]ethanol(80 g, 0.35 mol, 85% yield) as light yellow oil.

¹HNMR (400 MHz, CDCl₃) δ=7.43-7.35 (m, 6H), 7.25-7.18 (m, 6H), 7.18-7.11(m, 3H), 5.82 (d, J=3.8 Hz, 1H), 4.62 (t, J=4.2 Hz, 1H), 3.86 (td,J=3.5, 10.2 Hz, 1H), 3.61-3.47 (m, 2H), 3.37 (dd, J=2.8, 10.7 Hz, 1H),3.02 (dd, J=4.1, 10.7 Hz, 1H), 2.13 (tt, J=4.8, 9.9 Hz, 1H), 1.73-1.62(m, 2H), 1.42 (s, 3H), 1.40-1.31 (m, 1H), 1.26 (s, 3H). MS: [M+Na]⁺483.2

Step 7: Synthesis of(3aR,5S,6R,6aR)-6-(2-(benzyloxy)ethyl)-2,2-dimethyl-5-((trityloxy)methyl)tetrahydrofuro[2,3-d][1,3]dioxole

To a mixture of NaH (27.1 g, 0.68 mol, 60% in oil, 4.00 eq.) and THF(500 mL) is added dropwise a solution of2-[(3aR,5S,6S)-2,2-dimethyl-5-(trityloxymethyl)-3a,5,6,6a-tetrahydrofuro[2,3-d][1,3]dioxol-6-yl]ethanol(78 g, 0.17 mol) in THF (200 mL) at −20° C. over 5 min. After addition,the mixture is stirred at 25° C. for 2 h. BnBr (60.3 mL, 0.51 mol, 3.00eq.) is added dropwise. The mixture is stirred at 80° C. for 14 h. Aftercooling down to 0° C., the reaction is quenched by aq. NH₄Cl (sat., 20mL), diluted with H₂O (400 mL) and extracted with CH₂Cl₂ (400 mL×3). Thecombined organic layers are dried over Na₂SO₄, filtered, concentratedand purified by column chromatography (SiO₂, petroleum ether/ethylacetate=15:1 to 5:1) to give(3aR,5S,6S)-6-(2-benzyloxyethyl)-2,2-dimethyl-5-(trityloxymethyl)-3a,5,6,6a-tetrahydrofuro[2,3-d][1,3]dioxole(90 g, 0.16 mol, 97% yield) as white solid. (MS: [M+Na]=573.1).

Step 8: Synthesis of(3aR,5S,6R,6aR)-6-(2-(benzyloxy)ethyl)-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)methanol

To a solution of(3aR,5S,6S)-6-(2-benzyloxyethyl)-2,2-dimethyl-5-(trityloxymethyl)-3a,5,6,6a-tetrahydrofuro[2,3-d][1,3]dioxole(90 g, 0.16 mol) in CH₂Cl₂ (300 mL) is added CHCl₂COOH (30 mL, 0.16 mol,1.00 eq.). After 3 h at 25° C., the reaction mixture is neutralized withaq. NaHCO₃(sat., 500 mL) to pH˜7.0 at 0° C. The crude is extracted withCH₂Cl₂ (100 mL×3). The combined organic layers are dried over MgSO₄,filtered, concentrated and purified by column chromatography (SiO₂,petroleum ether/ethyl acetate=5:1 to 2:1) to give[(3aR,5S,6S)-6-(2-benzyloxyethyl)-2,2-dimethyl-3a,5,6,6a-tetrahydrofuro[2,3-d][1,3]dioxol-5-yl]methanol(44 g, 0.14 mol, 87% yield) as yellow oil.

Step 9: Synthesis of(3aR,5S,6R,6aR)-6-(2-(benzyloxy)ethyl)-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)methylbenzoate

To a solution of[(3aR,5S,6S)-6-(2-benzyloxyethyl)-2,2-dimethyl-3a,5,6,6a-tetrahydrofuro[2,3-d][1,3]dioxol-5-yl]methanol(62 g, 0.2 mol) in CH₂Cl₂ (200 mL) is added BzCl (35 mL, 0.3 mol, 1.50eq.) and Et₃N (55.7 mL, 0.4 mol, 2 eq.). After 1 h at 25° C., thereaction mixture is concentrated and purified by column chromatography(SiO₂, petroleum ether/ethyl acetate=15:1 to 10:1) to give[(3aR,5S,6S)-6-(2-benzyloxyethyl)-2,2-dimethyl-3a,5,6,6a-tetrahydrofuro[2,3-d][1,3]dioxol-5-yl]methylbenzoate (80 g, 0.19 mol, 97% yield) as light yellow oil. (MS: [M+Na]⁺435.1).

Step 10: Synthesis of((2S,3S,4R)-3-(2-(benzyloxy)ethyl)-4,5-dihydroxytetrahydrofuran-2-yl)methylbenzoate

To a mixture of[(3aR,5S,6S)-6-(2-benzyloxyethyl)-2,2-dimethyl-3a,5,6,6a-tetrahydrofuro[2,3-d][1,3]dioxol-5-yl]methylbenzoate (20 g, 49 mmol) and H₂O (6 mL) is added HOAc (28 mL, 10 eq.).The mixture is stirred at 100° C. for 5 h. After cooling down, thereaction mixture is neutralized with aq. NaHCO₃(sat., 2 L) and extractedwith CH₂Cl₂ (400 mL×3). The combined organic layers are concentrated andgive[(2S,3R,5R)-3-(2-benzyloxyethyl)-4,5-dihydroxy-tetrahydrofuran-2-yl]methylbenzoate (17.5 g, 47 mmol, 96% yield) as light yellow oil, which is usedfor the next step without purification. (MS: [M+Na]⁺ 395.1).

Step 11: Synthesis of(3R,4R,5S)-5-((benzoyloxy)methyl)-4-(2-(benzyloxy)ethyl)tetrahydrofuran-2,3-diyliacetate

To a solution of[(2S,3R,5R)-3-(2-benzyloxyethyl)-4,5-dihydroxy-tetrahydrofuran-2-yl]methylbenzoate (70 g, 0.19 mol) in pyridine (300 mL) is added Ac₂O (0.75 mol,70.4 mL, 4.0 eq.). The mixture is stirred at 60° C. for 4 h. Aftercooling to 25° C., the reaction mixture is neutralized with aq.NaHCO₃(sat.) to pH˜7 and extracted with CH₂Cl₂ (300 mL×3). The organiclayers are concentrated and purified by column chromatography (SiO₂,petroleum ether/ethyl acetate=10:1 to 5:1) to give[(2S,3S,5S)-4,5-diacetoxy-3-(2-benzyloxyethyl)tetrahydrofuran-2-yl]methylbenzoate (80 g, 93% yield) as white solid. (MS: [M+Na]+479.1).

Step 12: Synthesis of((2S,3R,4R,5R)-4-acetoxy-3-(2-(benzyloxy)ethyl)-5-(2-isobutyramido-6-oxo-1H-purin-9(6H)-yl)tetrahydrofuran-2-yl)methylbenzoate

To a solution of 2-methyl-N-(6-oxo-1,9-dihydropurin-2-yl)propanamide(18.9 g, 85.4 mmol, 1.30 eq.) in CH₃CN (300 mL) is added BSA (84.5 mL,341.7 mmol, 5.2 eq.) at 20° C. After stirring at 65° C. for 0.5 h, themixture is cooled down and concentrated. The residue is dissolved inMeCN (600 mL) followed by addition of a solution of[(2S,3S,5S)-4,5-diacetoxy-3-(2-benzyloxyethyl)tetrahydrofuran-2-yl]methylbenzoate (30 g, 65.7 mmol) in MeCN (150 mL) and TMSOTf (17.8 mL, 98.6mmol, 1.5 eq.) at −15° C. The mixture is stirred at 65° C. for 15 h.After cooling down, the mixture is concentrated and purified by columnchromatography (SiO₂, petroleum ether/ethyl acetate=5:1 to 1:1) to give((2S,3R,4R,5R)-4-acetoxy-3-(2-(benzyloxy)ethyl)-5-(2-isobutyramido-6-oxo-1H-purin-9(6H)-yl)tetrahydrofuran-2-yl)methylbenzoate (30 g, 48.6 mmol, 74% yield) as white solid. (MS: [M+1]⁺618.1).

¹HNMR (400 MHz, CHLOROFORM-d) δ=12.00 (s, 1H), 9.11 (s, 1H), 7.92-7.84(m, 2H), 7.82-7.76 (m, 1H), 7.58 (t, J=7.1 Hz, 1H), 7.46-7.37 (m, 2H),7.27-7.16 (m, 5H), 5.90-5.85 (m, 1H), 5.74 (d, J=5.3 Hz, 1H), 4.78-4.61(m, 2H), 4.55-4.38 (m, 3H), 3.55 (t, J=5.8 Hz, 2H), 3.23-3.14 (m, 1H),2.47 (spt, J=6.9 Hz, 1H), 2.22-2.10 (m, 3H), 1.83 (q, J=6.1 Hz, 2H),1.17 (dd, J=6.9, 8.9 Hz, 6H).

Step 13: Synthesis of((2S,3R,4R,5R)-4-acetoxy-3-(2-hydroxyethyl)-5-(2-isobutyramido-6-oxo-1H-purin-9(6H)-yl)tetrahydrofuran-2-yl)methylbenzoate

To a solution of[(2S,3S,5R)-4-acetoxy-3-(2-benzyloxyethyl)-5-[2-(2-methylpropanoylamino)-6-oxo-1H-purin-9-yl]tetrahydrofuran-2-yl]methylbenzoate (25 g, 40.5 mmol) in EtOH (500 mL) is added Pd/C (38 g, 10% oncarbon) and HOAc (25.00 mL, 437.1 mmol, 11 eq.) under N₂. The suspensionis purged with H₂ for 3 times and stirred under H₂ (40 Psi) for 48 h at50° C. After cooling down, the reaction mixture is filtered. Thefiltrate is concentrated and purified by column chromatography (SiO₂,petroleum ether/ethyl acetate=5:1 to 2:1) to give[(2S,3S,5R)-4-acetoxy-3-(2-hydroxyethyl)-5-[2-(2-methylpropanoylamino)-6-oxo-1H-purin-9-yl]tetrahydrofuran-2-yl]methylbenzoate (20 g, 37.9 mmol, 94% yield) as white solid. (MS: [M+1]⁺528.3).

Step 14: Synthesis of[(2S,3R,5R)-4-acetoxy-3-(2-iodoethyl)-5-[2-(2-methylpropanoylamino)-6-oxo-1H-purin-9-yl]tetrahydrofuran-2-yl]methylbenzoate

To a solution of[(2S,3R,5R)-4-acetoxy-3-(2-hydroxyethyl)-5-[2-(2-methylpropanoylamino)-6-oxo-1H-purin-9-yl]tetrahydrofuran-2-yl]methylbenzoate (3 g, 5.69 mmol, 1 eq.) in THF (90 mL) is added imidazole (1.16g, 17.06 mmol, 3 eq.) and triphenylphosphine (4.47 g, 17.06 mmol, 3 eq.)in one portion, then a solution of 12 (2.60 g, 10.24 mmol, 1.8 eq.) inTHF (10 mL) is added slowly. The reaction mixture is stirred at 25° C.for 16 h, quenched with saturated Na₂SO₃ aq. solution (8 mL) andevaporated to give the residue. The residue is dissolved in ethylacetate (80 mL) and washed by water (80 mL). The aqueous layer isextracted with EtOAc (150 mL×3). The combined organic layers are driedover anhydrous Na₂SO₄ and concentrated under reduced pressure. Theresidue is purified by column chromatography (SiO₂, petroleumether/ethyl acetate=1:1 to 1:3) to afford[(2S,3R,5R)-4-acetoxy-3-(2-iodoethyl)-5-[2-(2-methylpropanoylamino)-6-oxo-1H-purin-9-yl]tetrahydrofuran-2-yl]methylbenzoate (2.3 g, 64% yield) as yellow solid. (MS: [M+1]⁺ 638.2).

Step 15: Synthesis of[(2S,3R,4R,5R)-4-acetoxy-3-(2-azidoethyl)-5-[2-(2-methylpropanoylamino)-6-oxo-1H-purin-9-yl]tetrahydrofuran-2-yl]methylbenzoate

To a solution of[(2S,3R,4R,5R)-4-acetoxy-3-(2-iodoethyl)-5-[2-(2-methylpropanoylamino)-6-oxo-1H-purin-9-yl]tetrahydrofuran-2-yl]methylbenzoate (3.8 g, 5.96 mmol, 1 eq.) in THF (40 mL) is added NaN₃ (2.52 g,38.75 mmol, 6.5 eq.) and H₂O (10 mL). The mixture is stirred at 50° C.for 2 h. The mixture is quenched with saturated aq. Na₂CO₃ solution (50mL), and extracted with EtOAc (100 mL×3). The combined organic layersare dried over anhydrous Na₂SO₄ and concentrated under reduced pressureto afford[(2S,3R,4R,5R)-4-acetoxy-3-(2-azidoethyl)-5-[2-(2-methylpropanoylamino)-6-oxo-1H-purin-9-yl]tetrahydrofuran-2-yl]methylbenzoate (2 g, 61% yield) as yellow solid. (MS: [M+1]⁺ 553.1).

Step 16: Synthesis ofN-[9-[(2R,3R,4S,5S)-4-(2-azidoethyl)-3-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl]-6-oxo-1H-purin-2-yl]-2-methyl-propanamide

To a solution of[(2S,3R,5R)-4-acetoxy-3-(2-azidoethyl)-5-[2-(2-methylpropanoylamino)-6-oxo-1H-purin-9-yl]tetrahydrofuran-2-yl]methylbenzoate (2.55 g, 4.62 mmol, 1 eq.) in EtOH (220 mL) is added aq. NaOH(2M, 23 mL, 10 eq.) at 0° C. The resulting mixture is stirred at ambienttemperature for 0.5 h. To the reaction mixture is added HCOOH to adjustpH=7˜8 at 0° C., and the mixture is concentrated under reduced pressureto give a residue. The residue is purified by prep-HPLC (0.1% FA in MeCNand water, 0%˜70%) to affordN-[9-[(2R,3R,4S,5S)-4-(2-azidoethyl)-3-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl]-6-oxo-1H-purin-2-yl]-2-methyl-propanamide(1.6 g, 3.94 mmol, 85% yield) as white solid. (MS: [M+1]⁺ 407.1).

Step 17: Synthesis ofN-[9-[(2R,3R,4S,5S)-4-(2-azidoethyl)-5-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-3-hydroxy-tetrahydrofuran-2-yl]-6-oxo-1H-purin-2-yl]-2-methyl-propanamide

To a solution ofN-[9-[(2R,3R,4S,5S)-4-(2-azidoethyl)-3-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl]-6-oxo-1H-purin-2-yl]-2-methyl-propanamide (1.6 g, 3.94mmol) in pyridine (15 mL) is added DMTCl (1.60 g, 4.72 mmol, 1.2 eq.).The mixture is stirred at 25° C. for 3 h. The reaction mixture isquenched by addition of MeOH (10 mL) at 25° C. The mixture isconcentrated under reduced pressure to give a residue as yellow oil. Theresidue is purified by column chromatography (SiO₂, petroleumether/ethyl acetate=1/1 to EtOH/ethyl acetate=1:250) to giveN-[9-[(2R,3R,4S,5S)-4-(2-azidoethyl)-5-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-3-hydroxy-tetrahydrofuran-2-yl]-6-oxo-1H-purin-2-yl]-2-methyl-propanamide(950 mg, 1.34 mmol, 34% yield) as yellow solid. (MS: [M+1]⁺ 709.4).

Step 18: Synthesis of[(2R,3R,4R,5S)-4-(2-azidoethyl)-5-(hydroxymethyl)-2-[2-(2-methylpropanoylamino)-6-oxo-1H-purin-9-yl]tetrahydrofuran-3-yl]oxyphosphonicacid

To a solution ofN-[9-[(2R,3R,4S,5S)-4-(2-azidoethyl)-5-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-3-hydroxytetrahydrofuran-2-yl]-6-oxo-1H-purin-2-yl]-2-methyl-propanamide(700 mg, 988 μmol) in pyridine (6 mL) is added diphenyl phosphite (809.5mg, 3.46 mmol, 664 μL, 3.5 eq.). After 1 h at 25° C., DCM (5 mL) andEt₃N (3 mL) are added and the mixture is stirred at 25° C. for another1.5 h. The mixture is concentrated and the residue is partitionedbetween DCM (20 mL) and aq. NaHCO₃solution (5%, 20 mL). The organicphase is evaporated to give the crude intermediate, which isre-dissolved in a mixture of H₂O (3 mL) and 2,2-dichloroacetic acid (382mg, 2.96 mmol, 243 μL, 3 eq.) in DCM (10 mL). The mixture is stirred at25° C. for 0.5 h. The reaction is quenched with Et₃N (3.0 mL), then themixture is evaporated to give the residue. The residue is purified byreversed-phase column (0.1% TEA in MeCN and water, 0%˜70%) to give[(2R,3R,4R,5S)-4-(2-azidoethyl)-5-(hydroxymethyl)-2-[2-(2-methylpropanoylamino)-6-oxo-1H-purin-9-yl]tetrahydrofuran-3-yl]oxyphosphonicacid (400 mg, 82% yield, 95% purity) as white solid. (MS: [M+1]⁺ 471.0).

Synthesis of CDN-A from Intermediate 19

Step 1: Synthesis of[(2R,3R,4R,5R)-2-[[[(2R,3R,4R,5S)-4-(2-azidoethyl)-5-(hydroxymethyl)-2-[2-(2-methylpropanoylamino)-6-oxo-1H-purin-9-yl]tetrahydrofuran-3-yl]oxy-(2-cyanoethoxy)phosphoryl]oxymethyl]-5-(6-benzamidopurin-9-yl)-4-[tertbutyl(dimethyl)silyl]oxy-tetrahydrofuran-3-yl]oxyphosphonicacid

To a solution of[(2R,3R,4R,5R)-5-(6-benzamidopurin-9-yl)-4-[tert-butyl(dimethyl)silyl]oxy-2-(hydroxymethyl)tetrahydrofuran-3-yl]oxyphosphonicacid (400 mg, 727.8 mol) in CH₃CN (3 mL) is added tetrazole solution(0.45M in MeCN, 6.47 mL, 4 eq.). The mixture is stirred at 25° C. for 5min.N-[9-[(2R,3R,4R,5S)-4-(2-azidoethyl)-5-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-3-[2-cyanoethoxy-(diisopropylamino)phosphanyl]oxytetrahydrofuran-2-yl]-6-oxo-1H-purin-2-yl]-2-methyl-propanamide(595 mg, 655 μmol, 0.9 eq.) is added. After 0.5 h at 25° C.,2-hydroperoxy-2-methyl-propane (197 mg, 2.18 mmol, 209 μL, 3 eq.) isadded and the mixture is stirred at 25° C. for 0.5 h. Then2,2-dichloroacetic acid (938 mg, 7.28 mmol, 598 μL, 10 eq.) in DCM (10mL) is added. The mixture is stirred at 25° C. for 25 min and quenchedwith sat. aq. Na₂SO₃ (2.0 mL) followed by pyridine (2.0 mL) forneutralization. The mixture is evaporated to give the residue. Theresidue is purified by reversed-phase column (0.1% TEA in MeCN andwater, 0%˜70%) to afford[(2R,3R,4R,5R)-2-[[[(2R,3R,4R,5S)-4-(2-azidoethyl)-5-(hydroxyl-methyl)-2-[2-(2-methylpropanoylamino)-6-oxo-1H-purin-9-yl]tetrahydrofuran-3-yl]oxy-(2-cyanoethoxy)phosphoryl]oxymethyl]-5-(6-benzamidopurin-9-yl)-4-[tertbutyl(dimethyl)silyl]oxy-tetrahydrofuran-3-yl]oxyphosphinicacid (600 mg, 62% yield, 80% purity) as white solid. (MS: [M+1]⁺1071.5).

Step 2: Synthesis ofN-[9-[(27S,28R,29R,30R,31R,32R,33R,34R)-29-(2-azidoethyl)-32-[tert-butyl(dimethyl)silyl]oxy-67,68-dihydroxy-33-[2-(2-methylpropanoylamino)-6-oxo-1H-purin-9-yl]-67,68-dioxo-59,60,61,62,63,64-hexaoxa-67,68-diphosphatricyclooctadecan-34-yl]purin-6-yl]benzamide

To a solution of[(2R,3R,4R,5S)-4-(2-azidoethyl)-5-[[[(2R,3R,4R,5R)-5-(6-benzamidopurin-9-yl)-4-[tertbutyl(dimethyl)silyl]oxy-2-(hydroxymethyl)tetrahydrofuran-3-yl]oxy-(2-cyanoethoxy)phosphoryl]oxymethyl]-2-[2-(2-methylpropanoylamino)-6-oxo-1H-purin-9-yl]tetrahydrofuran-3-yl]oxyphosphinicacid (400 mg, 373.48 μmol) in pyridine (9 mL) is added2-chloro-5,5-dimethyl-1,3,2-dioxaphosphinane 2-oxide (345 mg, 1.87 mmol,5 eq.). After 15 min at 25° C., 12 (379 mg, 1.49 mmol, 4 eq.) and H₂O(13.5 mg, 747.0 μmol, 13.5 μL, 2 eq.) are added and the mixture isstirred at 25° C. for 0.5 h. The reaction is quenched with saturated aq.NaHCO₃solution (2.0 mL) and saturated aq. Na₂SO₃ solution (2.0 mL).After evaporation, the residue is dissolved in CH₃CN (10 mL) and addedwith 2-methylpropan-2-amine (10 mL). The mixture is stirred at 25° C.for 1 h. The reaction mixture is evaporated to give a residue. Theresidue is purified by reversed-phase column (0.1% TEA in MeCN andwater, 0%˜70%) to affordN-[9-[(27S,28R,29R,30R,31R,32R,33R,34R)-29-(2-azidoethyl)-32-[tert-butyl(dimethyl)silyl]oxy-67,68-dihydroxy-33-[2-(2-methylpropanoylamino)-6-oxo-1H-purin-9-yl]-67,68-dioxo-59,60,61,62,63,64-hexaoxa-67,68diphosphatricyclooctadecan-34-yl]purin-6-yl]benzamide(350 mg, 310 μmol, 83% yield, 90% purity) as white solid. (MS: [M+1]⁺1016.4).

Step 3: Synthesis of2-amino-9-[(19S,20R,21R,22R,23R,24R,25R,26R)-26-(6-aminopurin-9-yl)-21-(2-azidoethyl)-24-[tert-butyl(dimethyl)silyl]oxy-54,55-dihydroxy-54,55-dioxo-46,47,48,49,50,51-hexaoxa-54,55-diphosphatricyclooctadecan-25-yl]-1H-purin-6-one

N-[9-[(27S,28R,29R,30R,31R,32R,33R,34R)-29-(2-azidoethyl)-32-[tert-butyl(dimethyl)silyl]oxy-67,68-dihydroxy-33-[2-(2-methylpropanoylamino)-6-oxo-1H-purin-9-yl]-67,68-dioxo-59,60,61,62,63,64-hexaoxa-67,68diphosphatricyclooctadecan-34-yl]purin-6-yl]benzamide(300 mg, 295 μmol) is dissolved in MeNH₂/EtOH (5M, 2.95 mL) and themixture is stirred at 25° C. for 2 h. The mixture is evaporated to givea residue. The residue is purified by reversed-phase column (0.1% TEA inMeCN and water, 0%˜35%) to afford2-amino-9-[(19S,20R,21R,22R,23R,24R,25R,26R)-26-(6-aminopurin-9-yl)-21-(2-azidoethyl)-24-[tert-butyl(dimethyl)silyl]oxy-54,55-dihydroxy-54,55-dioxo-46,47,48,49,50,51-hexaoxa-54,55diphosphatricyclooctadecan-25-yl]-1H-purin-6-one(160 mg, 52% yield, 80% purity) as white solid. (MS: [M+1]⁺ 842.3).

Step 4: Synthesis of2-amino-9-[(19S,20R,21R,22R,23R,24R,25R,26R)-21-(2-aminoethyl)-26-(6-aminopurin-9-yl)-24-[tert-butyl(dimethyl)silyl]oxy-52,53-dihydroxy-52,53-dioxo-44,45,46,47,48,49-hexaoxa-52,53-diphosphatricyclooctadecan-25-yl]-1H-purin-6-one

To the solution of2-amino-9-[(19S,20R,21R,22R,23R,24R,25R,26R)-26-(6-aminopurin-9-yl)-21-(2-azidoethyl)-24-[tertbutyl(dimethyl)silyl]oxy-54,55-dihydroxy-54,55-dioxo-46,47,48,49,50,51-hexaoxa-54,55diphosphatricyclooctadecan-25-yl]-1Hpurin-6-one(100 mg, 118.8 μmol) in MeOH (8 mL) is added Pd/C (30 mg, 10% purity) inone portion, and the mixture is stirred at 25° C. under H₂ (10 psi) for5 h. The mixture is filtered and the filtrate is evaporated to give aresidue. The residue is purified by reversed-phase column (0.1% TEA inMeCN and water, 0%˜30%) to afford2-amino-9-[(19S,20R,21R,22R,23R,24R,25R,26R)-21-(2-aminoethyl)-26-(6-aminopurin-9-yl)-24-[tert-butyl(dimethyl)silyl]oxy-52,53-dihydroxy-52,53-dioxo-44,45,46,47,48,49-hexaoxa-52,53diphosphatricyclooctadecan-25-yl]-1H-purin-6-one(70 mg, 77.2 μmol, 65% yield, 90% purity) as white solid. (MS: [M+1]⁺816.5).

Step 5: Synthesis of2-amino-9-[(14S,15R,16R,17R,18S,19R,20R,21R)-16-(2-aminoethyl)-20-(6-aminopurin-9-yl)-17,45,46-trihydroxy-45,46-dioxo-39,40,41,42,43,44-hexaoxa-45,46-diphosphatricyclooctadecan-21-yl]-1H-purin-6-one(CDN-A)

To the solution of2-amino-9-[(19S,20R,21R,22R,23R,24R,25R,26R)-21-(2-aminoethyl)-26-(6-aminopurin-9-yl)-24-[tertbutyl(dimethyl)silyl]oxy-52,53-dihydroxy-52,53-dioxo-44,45,46,47,48,49-hexaoxa-52,53diphosphatricyclooctadecan-25-yl]-1Hpurin-6-one(35 mg, 42.9 μmol) in MeOH (3 mL) is added NH₄F (127 mg, 3.43 mmol, 80eq.) in one portion, and the mixture is stirred at 70° C. for 0.25 h.After cooling down to room temperature, the mixture is evaporated togive a residue. The residue is purified by reversed-phase column (0.1%HCOOH in MeCN and water, 0%˜30%) to afford2-amino-9-[(14S,15R,16R,17R,18S,19R,20R,21R)-16-(2-aminoethyl)-20-(6-aminopurin-9-yl)-17,45,46-trihydroxy-45,46-dioxo-39,40,41,42,43,44-hexaoxa-45,46diphosphatricyclooctadecan-21-yl]-1H-purin-6-one(CDN-A, 8.6 mg, 11.9 μmol, 28% yield, 97% purity) as white solid. (MS:[M+1]⁺ 702.0).

¹H NMR (D₂O+buffer, 400 MHz): δ (ppm) 8.19 (s, 1H), 8.18 (s, 1H), 7.76(s, 1H), 6.08 (s, 1H), 5.83-5.81 (m, 1H), 5.71-5.67 (m, 1H), 5.03-5.01(m, 1H), 4.42-4.37 (m, 2H), 4.30-4.28 (m, 1H), 4.18-4.16 (m, 1H),4.07-4.01 (m, 2H), 3.13-3.11 (m, 2H), 2.68-2.66 (m, 1H), 2.33-2.30 (m,1H), 1.85-1.83 (m, 1H). ³¹P NMR (D₂O+buffer): δ (ppm) −1.15, −2.47

Example 2. Preparation of CDN-B

Schemes B1 and B2 below depict the synthesis of a CDN (“CDN-B”)disclosed herein. The synthesis and characterization of this CDN and thesynthetic intermediates employed are described below.

Synthesis of Intermediate 29 from Intermediate 14

Step 1: Synthesis of[(2S,3R,4R,5R)-4-acetoxy-3-(2-chloroethyl)-5-[2-(2-methylpropanoylamino)-6-oxo-1H-purin-9-yl]tetrahydrofuran-2-yl]methylbenzoate

To a solution of[(2S,3R,4R,5R)-4-acetoxy-3-(2-hydroxyethyl)-5-[2-(2-methylpropanoylamino)-6-oxo-1H-purin-9-yl]tetrahydrofuran-2-yl]methylbenzoate (19 g, 36.0 mmol) in DMF (100 mL) is added PPh₃ (23.6 g, 90.0mmol, 2.5 eq.) and CCl₄ (17.3 mL, 180.1 mmol, 5 eq.). After beingstirred for 16 h at 25° C., the reaction is quenched by sat. aq.NaHCO₃(150 mL) and extracted with EtOAc (80 mL×2). The combined organiclayers are concentrated and purified by column chromatography (SiO₂,petroleum ether/ethyl acetate=10:1 to 1:1) to give[(2S,3R,4R,5R)-4-acetoxy-3-(2-chloroethyl)-5-[2-(2-methylpropanoylamino)-6-oxo-1H-purin-9-yl]tetrahydrofuran-2-yl]methylbenzoate (13 g, 23.8 mmol, 66% yield) as light yellow solid. (MS: [M+1]⁺546.2).

Step 2: Synthesis of[(2S,3R,4R,5R)-4-acetoxy-3-(2-acetylsulfanylethyl)-5-[2-(2-methylpropanoylamino)-6-oxo-1H-purin-9-yl]tetrahydrofuran-2-yl]methylbenzoate

To a solution of[(2S,3R,4R,5R)-4-acetoxy-3-(2-chloroethyl)-5-[2-(2-methylpropanoylamino)-6-oxo-1H-purin-9-yl]tetrahydrofuran-2-yl]methylbenzoate (15 g, 27.5 mmol) in DMF (100 mL) is added AcSK (7.84 g, 68.7mmol, 2.5 eq.). The reaction mixture is stirred at 50° C. for 16 h.After cooling down, the reaction mixture is diluted with DCM (200 mL)and washed with aq. NaHCO₃(sat., 200 mL). The organic layer isconcentrated to give[(2S,3R,4R,5R)-4-acetoxy-3-(2-acetylsulfanylethyl)-5-[2-(2-methylpropanoylamino)-6-oxo-1H-purin-9-yl]tetrahydrofuran-2-yl]methylbenzoate (16 g) as light yellow oil which is used for the next stepwithout purification. (MS: [M+1]⁺ 586.3).

Step 3: Synthesis ofN-[9-[(2R,3R,4S,5S)-3-hydroxy-5-(hydroxymethyl)-4-(2-sulfanylethyl)tetrahydrofuran-2-yl]-6-oxo-1H-purin-2-yl]-2-methyl-propanamide

To a solution of[(2S,3R,4R,5R)-4-acetoxy-3-(2-acetylsulfanylethyl)-5-[2-(2-methylpropanoylamino)-6-oxo-1H-purin-9-yl]tetrahydrofuran-2-yl]methylbenzoate (16 g, 27.3 mmol) in EtOH (160 mL) is added NaOH (2 M, 68.3 mL,5 eq.) at 0° C. The reaction mixture is stirred at 0° C. for 0.5 h. ThepH is adjusted to 7 by HOAc. The mixture is concentrated in vacuum toremove most of solvent. The brown precipitate is collected and treatedwith DCM/TBME (1/100,V/V, 200 mL). After filtration, the filtrate isconcentrated to giveN-[9-[(2R,3R,4S,5S)-3-hydroxy-5-(hydroxymethyl)-4-(2-sulfanylethyl)tetrahydrofuran-2-yl]-6-oxo-1H-purin-2-yl]-2-methyl-propanamide(11 g, crude, ˜10% disulfide) as brown solid which is used for the nextstep without further purification. (MS: [M+1]⁺ 398.1).

Step 4: Synthesis ofN-[9-[(2R,3R,4S,5S)-3-hydroxy-5-[[(3-methoxyphenyl)-(4-methoxyphenyl)-phenyl-methoxy]methyl]-4-[2-[(3-methoxyphenyl)-(4-methoxyphenyl)-phenyl-methyl]sulfanylethyl]tetrahydrofuran-2-yl]-6-oxo-1H-purin-2-yl]-2-methyl-propanamide

To a solution ofN-[9-[(2R,3R,4S,5S)-3-hydroxy-5-(hydroxymethyl)-4-(2-sulfanylethyl)tetrahydrofuran-2-yl]-6-oxo-1H-purin-2-yl]-2-methyl-propanamide(11 g, 27.7 mmol) in py. (110 mL) is added DMTCl (28.1 g, 83.0 mmol, 3eq.). After 16 h at 25° C., the reaction is quenched with aq.NaHCO₃(sat., 200 mL) and extracted with EtOAc (200 mL×2). The organicphase is dried over Na₂SO₄, filtered and purified by silica gelchromatography PE:EE(EA:EtOH=3:1)=10:1˜2:1 to give compoundN-[9-[(2R,3R,4S,5S)-3-hydroxy-5-[[(3-methoxyphenyl)-(4-methoxyphenyl)-phenyl-methoxy]methyl]-4-[2-[(3-methoxyphenyl)-(4-methoxyphenyl)-phenyl-methyl]sulfanylethyl]tetrahydrofuran-2-yl]-6-oxo-1H-purin-2-yl]-2-methyl-propanamide(14 g, 47.0% yield, 93% purity) as light yellow solid. (MS: [M+1]⁺1002.5).

Step 5: Synthesis of[(2R,3R,4R,5S)-5-(hydroxymethyl)-4-[2-[(3-methoxyphenyl)-(4-methoxyphenyl)-phenyl-methyl]sulfanylethyl]-2-[2-(2-methylpropanoylamino)-6-oxo-1H-purin-9-yl]tetrahydrofuran-3-yl]oxyphosphinicacid

To a solution ofN-[9-[(2R,3R,4S,5S)-3-hydroxy-5-[[(3-methoxyphenyl)-(4-methoxyphenyl)-phenyl-methoxy]methyl]-4-[2-[(3-methoxyphenyl)-(4-methoxyphenyl)-phenyl-methyl]sulfanylethyl]tetrahydrofuran-2-yl]-6-oxo-1H-purin-2-yl]-2-methyl-propanamide(12 g, 8.4 mmol) in py. (120 mL) is added phenoxyphosphonoyloxybenzene(7.51 mL, 29.3 mmol, 3.5 eq.) at 25° C. After 1 h, Et₃N/H₂O (100 mL,1:1) is added. After 0.5 h, the mixture is extracted with EtOAc (200mL×2). The organic phase is concentrated and then dissolved in aqueousAcOH (80%, 120 mL). The mixture is stirred at 25° C. for 1 h. Thereaction mixture is neutralized by addition of saturated aq. Na₂CO₃ at0° C. till pH˜7. The mixture is directly purified by reverse phasecolumn (CH₃CN/H₂O, 0˜60%) and give[(2R,3R,4R,5S)-5-(hydroxymethyl)-4-[2-[(3-methoxyphenyl)-(4-methoxyphenyl)-phenyl-methyl]sulfanylethyl]-2-[2-(2-methylpropanoylamino)-6-oxo-1H-purin-9-yl]tetrahydrofuran-3-yl]oxyphosphinicacid (4.2 g, 5.5 mmol, 65% yield) as a white solid. (MS: [M+1]⁺ 764.4).

Synthesis of CDN-B from Intermediate 29

Step 1: Synthesis of[(2R,3R,4R,5S)-5-[[[(2R,3R,4R,5R)-5-(6-benzamidopurin-9-yl)-4-[tert-butyl(dimethyl)silyl]oxy-2-(hydroxymethyl)tetrahydrofuran-3-yl]oxy-(2-cyanoethoxy)phosphoryl]oxymethyl]-4-[2-[bis(4-methoxyphenyl)-phenyl-methyl]sulfanylethyl]-2-[2-(2-methylpropanoylamino)-6-oxo-1H-purin-9-yl]tetrahydrofuran-3-yl]oxyphosphinicacid

To a solution of[(2R,5S)-4-[2-[bis(4-methoxyphenyl)-phenyl-methyl]sulfanylethyl]-5-(hydroxymethyl)-2-[2-(2-methylpropanoylamino)-6-oxo-1H-purin-9-yl]tetrahydrofuran-3-yl]oxyphosphinicacid (0.75 g, 0.98 mmol) in 1H-tetrazole (0.45 M in MeCN, 22.50 mL, 10eq.) is addedN-[9-[(2R,3R,4R,5R)-5-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-3-[tert-butyl(dimethyl)silyl]oxy-4-[2-cyanoethoxy-(diisopropylamino)phosphanyl]oxy-tetrahydrofuran-2-yl]purin-6-yl]benzamide(1.1 g, 1.1 mmol, 1.1 eq.). After 1 h, TBHP (0.43 ml, 65% in decane, 3eq.) is added. The reaction mixture is stirred at 25° C. for 0.5 h. Thereaction is quenched by aq. sodium bisulfite solution (33%, 4 mL) at 0°C. and extracted with EtOAc (100 mL×2). The organic phase isconcentrated and dissolved in aq. AcOH (80%, 20 mL). After 1 h, thereaction mixture is neutralized by aq. Na₂CO₃ (sat.) at 0° C. Themixture is purified by reverse phase column (CH₃CN/H₂O, neutralcondition, 0˜60%) to give[(2R,3R,4R,5S)-5-[[[(2R,3R,4R,5R)-5-(6-benzamidopurin-9-yl)-4-[tert-butyl(dimethyl)silyl]oxy-2-(hydroxymethyl)tetrahydrofuran-3-yl]oxy-(2-cyanoethoxy)phosphoryl]oxymethyl]-4-[2-[bis(4-methoxyphenyl)-phenyl-methyl]sulfanylethyl]-2-[2-(2-methylpropanoylamino)-6-oxo-1H-purin-9-yl]tetrahydrofuran-3-yl]oxyphosphinicacid (0.79 g, 48% yield, 80% purity) as white solid. (MS: [M+1]⁺1364.0).

Step 2: Synthesis ofN-[9-[(47S,48R,49R,50R,51R,52R,53R,54R)-49-[2-[bis(4-methoxyphenyl)-phenyl-methyl]sulfanylethyl]-52-[tert-butyl(dimethyl)silyl]oxy-87,88-dihydroxy-53-[2-(2-methylpropanoylamino)-6-oxo-1H-purin-9-yl]-87,88-dioxo-77,78,79,80,81,82-hexaoxa-87,88-diphosphatricyclooctadecan-54-yl]purin-6-yl]benzamide

To a solution of[(2R,3R,4R,5S)-5-[[[(2R,3R,4R,5R)-5-(6-benzamidopurin-9-yl)-4-[tert-butyl(dimethyl)silyl]oxy-2-(hydroxymethyl)tetrahydrofuran-3-yl]oxy-(2-cyanoethoxy)phosphoryl]oxymethyl]-4-[2-[bis(4-methoxyphenyl)-phenyl-methyl]sulfanylethyl]-2-[2-(2-methylpropanoylamino)-6-oxo-1H-purin-9-yl]tetrahydrofuran-3-yl]oxyphosphinicacid (0.79 g, 0.58 mmol) in pyridine (16 mL) is added2-chloro-5,5-dimethyl-1,3,2-dioxaphosphinane 2-oxide (0.7 g, 3.8 mmol,6.5 eq.). After 0.5 h, CCl₄ (3.2 g, 20.6 mmol, 1.98 mL, 35.5 eq.), H₂O(0.16 mL, 8.79 mmol) and NMM (0.79 mL) is added. The reaction mixture isstirred at 25° C. for 0.5 h before pouring into aq. NaHSO₃ (sat., 10mL). After 5 min, aq. NaHCO₃(sat., 20 mL) is added slowly. The mixtureis extracted with EtOAc (30 mL×2). The organic phase is concentrated anddissolved in CH₃CN (8 mL) and t-BuNH₂ (8 mL). After 0.5 h, the mixtureis concentrated and purified by reverse phase column (CH₃CN/H₂O, neutralcondition, 0˜40%) to giveN-[9-[(47S,48R,49R,50R,51R,52R,53R,54R)-49-[2-[bis(4-methoxyphenyl)-phenyl-methyl]sulfanylethyl]-52-[tert-butyl(dimethyl)silyl]oxy-87,88-dihydroxy-53-[2-(2-methylpropanoylamino)-6-oxo-1H-purin-9-yl]-87,88-dioxo-77,78,79,80,81,82-hexaoxa-87,88diphosphatricyclooctadecan-54-yl]purin-6-yl]benzamide(0.5 g, 60% yield, 90.8% purity) as white solid. (MS: [M+1]⁺ 1309.8).

Step 3: Synthesis of2-amino-9-[(39S,40R,41R,42R,43R,44R,45R,46R)-46-(6-aminopurin-9-yl)-41-[2-[bis(4-methoxyphenyl)-phenyl-methyl]sulfanylethyl]-44-[tert-butyl(dimethyl)silyl]oxy-74,75-dihydroxy-74,75-dioxo-64,65,66,67,68,69-hexaoxa-74,75-diphosphatricyclooctadecan-45-yl]-1H-purin-6-one

The mixture ofN-[9-[(47S,48R,49R,50R,51R,52R,53R,54R)-49-[2-[bis(4-methoxyphenyl)-phenyl-methyl]sulfanylethyl]-52-[tert-butyl(dimethyl)silyl]oxy-87,88-dihydroxy-53-[2-(2-methylpropanoylamino)-6-oxo-1H-purin-9-yl]-87,88-dioxo-77,78,79,80,81,82-hexaoxa-87,88diphosphatricyclooctadecan-54-yl]purin-6-yl]benzamide(500 mg, 0.38 mmol) and methylamine alcohol solution (10 mL, 30%) isstirred at 25° C. for 4 h. The reaction mixture is concentrated invacuum. The residue is purified by reverse phase column (CH₃CN/H₂O,neutral condition, 0˜30%) to give2-amino-9-[(39S,40R,41R,42R,43R,44R,45R,46R)-46-(6-aminopurin-9-yl)-41-[2-[bis(4-methoxyphenyl)-phenyl-methyl]sulfanylethyl]-44-[tert-butyl(dimethyl)silyl]oxy-74,75-dihydroxy-74,75-dioxo-64,65,66,67,68,69-hexaoxa-74,75diphosphatricyclooctadecan-45-yl]-1H-purin-6-one(220 mg, 48% yield, 95% purity) as white solid.

Step 4: Synthesis of:2-amino-9-[(19S,20R,21R,22R,23R,24R,25R,26R)-26-(6-aminopurin-9-yl)-24-[tert-butyl(dimethyl)silyl]oxy-51,52-dihydroxy-51,52-dioxo-21-(2-sulfanylethyl)-43,44,45,46,47,48-hexaoxa-51,52-diphosphatricyclooctadecan-25-yl]-1H-purin-6-one

To a solution of2-amino-9-[(39S,40R,41R,42R,43R,44R,45R,46R)-46-(6-aminopurin-9-yl)-41-[2-[bis(4-methoxyphenyl)-phenyl-methyl]sulfanylethyl]-44-[tert-butyl(dimethyl)silyl]oxy-74,75-dihydroxy-74,75-dioxo-64,65,66,67,68,69-hexaoxa-74,75diphosphatricyclooctadecan-45-yl]-1H-purin-6-one(190 mg, 0.17 mmol) in DCM (4 mL) is added 2,2-dichloroacetic acid (0.8mL, 9.74 mmol, 58 eq.). The mixture is stirred at 25° C. for 1 h andneutralized by water/Et₃N (1:1, V/V, 3 mL) at 0° C. The mixture isconcentrated and purified by reverse phase column (CH₃CN/H₂O, contains0.05% TEA, 0% to 40%) to give2-amino-9-[(19S,20R,21R,22R,23R,24R,25R,26R)-26-(6-aminopurin-9-yl)-24-[tert-butyl(dimethyl)silyl]oxy-51,52-dihydroxy-51,52-dioxo-21-(2-sulfanylethyl)-43,44,45,46,47,48-hexaoxa-51,52diphosphatricyclooctadecan-25-yl]-1H-purin-6-one(36 mg, 25% yield, 98% purity, TEA salt) as white solid. (MS: [M+1]⁺833.3).

Step 5: Synthesis of2-amino-9-[(14S,15R,16R,17R,18S,19R,20R,21R)-20-(6-aminopurin-9-yl)-17,44,45-trihydroxy-44,45-dioxo-16-(2-sulfanylethyl)-38,39,40,41,42,43-hexaoxa-44,45-diphosphatricyclooctadecan-21-yl]-1H-purin-6-one(CDN-B)

To a solution of2-amino-9-[(19S,20R,21R,22R,23R,24R,25R,26R)-26-(6-aminopurin-9-yl)-24-[tert-butyl(dimethyl)silyl]oxy-51,52-dihydroxy-51,52-dioxo-21-(2-sulfanylethyl)-43,44,45,46,47,48-hexaoxa-51,52diphosphatricyclooctadecan-25-yl]-1H-purin-6-one(36 mg, 0.043 mmol) in MeOH (4 mL) is added NH₄F (0.16 g, 4.32 mmol, 100eq.). The mixture is stirred at 70° C. for 1 h, concentrated andpurified by reverse phase column (CH₃CN/H₂O, contains 0.05% FA, 0% to30%) to give2-amino-9-[(14S,15R,16R,17R,18S,19R,20R,21R)-20-(6-aminopurin-9-yl)-17,44,45-trihydroxy-44,45-dioxo-16-(2-sulfanylethyl)-38,39,40,41,42,43-hexaoxa-44,45diphosphatricyclooctadecan-2l-yl]-1H-purin-6-one(CDN-B, 7 mg, 22.5% yield, 99.8% purity) as white solid.

¹H NMR (400 MHz, D₂O) δ=8.25 (s, 1H), 8.19 (s, 1H), 7.77 (s, 1H), 6.10(s, 1H), 5.80 (d, J=8.3 Hz, 1H), 5.67 (q, J=8.2 Hz, 1H), 5.05-4.98 (m,1H), 4.43 (d, J=9.0 Hz, 1H), 4.36 (d, J=12.1 Hz, 1H), 4.31 (brs, 1H),4.18 (d, J=11.7 Hz, 1H), 4.08-3.97 (m, 2H), 3.11 (q, J=7.2 Hz, 1H),2.79-2.66 (m, 2H), 2.62-2.51 (m, 1H), 2.28-2.13 (m, 1H), 1.84-1.73 (m,1H), 1.19 (t, J=7.3 Hz, 1H). ³¹P NMR: −0.951, −2.201. MS: [M+1]⁺ 718.9.

Example 3. Preparation of Target-Binding Antibodies

Anti-PD-L1 antibodies

Ab-A1 (mu-anti-PDL1): Expression vectors encoding a murine anti-humanPD-L1 antibody Ab-A1 (mu-anti-PDL1) having a heavy chain of SEQ ID NO:3and a light chain of SEQ ID NO:5 were prepared by cloning cDNAs encodinga variable heavy (VH) chain of SEQ ID NO:1 and variable light (VL) chainof SEQ ID NO:2 into separate pFUSEss-CHIg-mG2a (mouse IgG2a heavy chainconstant region) and pFUSE2ss-CLIg-mk (mouse kappa light chain constantregion) expression vectors, respectively. The VH and VL of Ab-A1 arebased on atezolizumab.

Ab-A2 (mu-anti-PDL1-cys): Expression vectors encoding a murineanti-human PD-L1 antibody Ab-A2 (mu-anti-PDL1-cys) having a heavy chainof SEQ ID NO:4 and a light chain of SEQ ID NO:5 were prepared usinganalogous techniques as Ab-A1. The VH and VL sequences of Ab-A2 arebased on atezolizumab. The heavy chain constant region of Ab-A2 has twomutations relative to wild-type mouse IgG2a, the first being a leucineto phenylalanine substitution at position 234 in the CH₂ domain (i.e.L234F, numbered in accordance to the wild-type mouse IgG2a sequencealigned with the human IgG1 sequence, using Eu numbering), and thesecond being a serine to cysteine substitution at position 239 in theCH₂ domain to present an additional cysteine for conjugation (i.e.S239C, using the same numbering).

The sequences of heavy chains of anti-human PD-L1 antibodies Ab-A1 andAb-A2 are shown below as SEQ ID NOS:3 and 4, respectively. The heavychain variable region is underlined in each sequence, which is the samefor both, and corresponds to SEQ ID NO:1. The L234F and S239C CH2 domainmutations in SEQ ID NO:4 for Ab-A2 are shown in bold.

Heavy chain for Ab-A1  (SEQ ID NO: 3)EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSAAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKS FSRTPGKHeavy chain for Ab-A2  (SEQ ID NO: 4)EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSAAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNFLGGPCVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKS FSRTPGK

The sequence of the light chains of anti-human PD-L1 antibodies Ab-A1and Ab-A2 is shown below as SEQ ID NO:5. The light chain variable regionis underlined in the sequence and corresponds to SEQ ID NO:2.

Light chain for Ab-A1 and Ab-A2  (SEQ ID NO: 5)DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEA THKTSTSPIVKSFNRNEC

Ab-A3 (rt-anti-PDL1): A rat anti-mouse PD-L1 antibody Ab-A3 having a ratIgG2b heavy chain constant region was sourced commercially from BioXcell(BE0101).

Anti-EGFR Antibodies

Ab-B1 (mu-anti-EGFR): A murine anti-human EGFR antibody Ab-B1 having amouse IgG2a heavy chain constant region was sourced commercially fromBioXcell (BE0279).

Ab-B2 (mu-anti-EGFR-cys): Expression vectors encoding a murineanti-human EGFR antibody Ab-B2 having a heavy chain of SEQ ID NO:8 and alight chain of SEQ ID NO:9 were prepared by cloning cDNAs encoding avariable heavy (VH) chain of SEQ ID NO:6 and variable light (VL) chainof SEQ ID NO:7 into separate pFUSEss-CHIg-mG2a (mouse IgG2a heavy chainconstant region) and pFUSE2ss-CLIg-mk (mouse kappa light chain constantregion) expression vectors, respectively. The VH and VL sequences ofAb-B2 are based on cetuximab. The CH2 domain of the heavy chain of Ab-B2features mutations L234F and S239C relative to wild-type mouse IgG2a, asdescribed previously for Ab-A2.

The sequence of the heavy chain of anti-human EGFR antibody Ab-B2 isshown below as SEQ ID NO:8. The heavy chain variable region isunderlined and corresponds to SEQ ID NO:6. The L234F and S239C mutationsin the CH2 domain are shown in bold.

Heavy chain for Ab-B2  (SEQ ID NO: 8)QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSAAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNFLGGPCVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTK SFSRTPGK

The sequence of the light chain of Ab-B2 is shown below as SEQ ID NO:9.The variable region is underlined and corresponds to SEQ ID NO:7.

Light chain for Ab-B2  (SEQ ID NO: 9)DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKT STSPIVKSFNRNEC

Ab-B3 (hu-anti-EGFR): Expression vectors encoding a murine-humanchimeric anti-human EGFR antibody Ab-B3 having a heavy chain of SEQ IDNO:12 and a light chain of SEQ ID NO:13 were prepared by cloning cDNAsencoding a variable heavy (VH) chain of SEQ ID NO:10 plus a human IgG1heavy chain constant region, and a variable light (VL) chain of SEQ IDNO:11 plus a human Ig kappa light chain constant region into separatepcDNA3.4 expression vectors. The full length heavy and light chainsequences of Ab-B2 are based on cetuximab.

The sequence of the heavy chain of anti-human EGFR antibody Ab-B3 isshown below as SEQ ID NO:12. The heavy chain variable region isunderlined and corresponds to SEQ ID NO:10.

Heavy chain for Ab-B3  (SEQ ID NO: 12)QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGK

The sequence of the light chain of Ab-B3 is shown below as SEQ ID NO:13.The variable region is underlined and corresponds to SEQ ID NO:11.

Light chain for Ab-B3  (SEQ ID NO: 13)DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC

Ab-B4 (hu-anti-EGFR-A/C,V/C): An expression vector encoding a humananti-human EGFR antibody Ab-B4 having a heavy chain of SEQ ID NO:16 anda light chain of SEQ ID NO:17 was prepared by cloning cDNAs encoding avariable heavy (VH) chain of SEQ ID NO:14 plus a human IgG1 heavy chainconstant region, and a variable light (VL) chain of SEQ ID NO:15 plus amodified human Ig kappa light chain constant region into separatepcDNA3.4 expression vectors. The VH and VL of Ab-B4 are based oncetuximab, with VH having an alanine to cysteine mutation at position109 adjacent to the CH1 domain (i.e., A109C). The light chain of Ab-B4features a valine to cysteine mutation at position 205 (i.e., V205C).

The sequence of the heavy chain of anti-human EGFR antibody Ab-B4 isshown below as SEQ ID NO:16. The heavy chain variable region isunderlined and corresponds to SEQ ID NO:14. The A109C mutation is shownin bold.

Heavy chain for Ab-B4  (SEQ ID NO: 16)QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARA LTYYDYEFAYWGQGTLVTVSCASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGK

The sequence of the light chain of Ab-B4 is shown below as SEQ ID NO:17.The variable region is underlined and corresponds to SEQ ID NO:15. TheV205C mutation is shown in bold.

Light chain for Ab-B4  (SEQ ID NO: 17)DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPCTKSFNRGECAnti-HER2 Antibodies

Ab-C1 (anti-HER2): A humanized anti-human HER2 antibody Ab-C1 wassourced commercially as trastuzumab (Herceptin®).

The sequence of the heavy chain of anti-human HER2 antibody Ab-C1 isshown below as SEQ ID NO:20. The heavy chain variable region isunderlined and corresponds to SEQ ID NO:18.

Heavy chain for Ab-C1  (SEQ ID NO: 20)EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPGK

The sequence of the light chain of Ab-C1 is shown below as SEQ ID NO:21.The variable region is underlined and corresponds to SEQ ID NO:19.

Light chain for Ab-C1  (SEQ ID NO: 21)DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGECAnti-CD47 Antibodies

Ab-D1 (rt-anti-CD47): A rat anti-mouse CD47 antibody Ab-D1 having a ratIgG2b heavy chain constant region was sourced commercially from BioXcell(BE0270).

Expression and Purification

Plasmids encoding the heavy chain and light chain of target-bindingantibodies were transfected into CHO cells for expression of antibodiesusing ExpiFectamine™ CHO Transfection Kit (ThermoFisher Scientific, CatNo: A29129) according to manufacturer's protocol. Total amount ofplasmids used for transfection was 0.5 ug/ml CHO cell, with a ratiobetween heavy chain and light chain plasmids of 2:3. Six days aftertransfection, CHO cells were spun down and the media filtered, thenloaded onto protein A beads (HiTrap Protein A HP, GE, Cat No:17-0403-01) and eluted with 0.1 M Glycine (pH 3.0). The eluted antibodyfractions were pooled and concentrated to 1 ml and buffer exchanged intoPBS by size exclusion (ENrich Sec650, Bio-Rad, Cat No: 780-1650) beforestorage at −80° C.

Example 4. Preparation of CDN-A ADCs

Synthesis of CDN-A-Linker

CDN-A (27 mg, 0.0385 mmol) is co-evaporated with Py. (3×3 ml) and driedunder high vacuum before use.4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl(4-nitrophenyl) carbonate (54 mg, 0.0732 mmol, 2.0 eq.), HOBt (5.2 mg,0.0385 mmol, 1.0 eq.) and DMF (2.5 ml) are added. With stirring, DIPEA(67 μl, 0.385 mmol, 10 eq.) is added. The mixture is stirred at roomtemperature under N₂ for 42 hours. The reaction mixture is diluted by asolvent mixture of ethyl acetate (25 ml), t-butyl methyl ether (75 ml)and acetic acid (30 μL, 0.521 mmol). The solid is collected bycentrifugation and washing with t-butyl methyl ether (3×75 ml) to give44 mg of crude. 20 mg of the crude is further purified by reverse phasecolumn (CH₃CN/H₂O, 0.10% FA, 0% to 30%) to give desired product (7 mg,31% yield, 97% pure). (MS: [M−1]⁻ 1298.2).Conjugation of Antibodies to CDN-A

Reduction of antibody disulfides: Target-binding antibodies were firstreduced with Tris-(2-carboxyethyl)-phosphine hydrochloride (TCEP) (2.3molar equivalents of TCEP at room temperature for 1.5 hours, or 40 molarequivalents of TCEP at 37° C. for 2 hours. Excess TCEP was removed usinga desalting column (HiTrap Desalting column, GE, Cat. No: 29048684).

Re-oxidation of hinge cysteines: The hinge cysteines of the reducedantibody were re-oxidized with 200 molar equivalent of dehydroascorbicacid (DHA, 0.5 M in DMSO) at 37° C. for 4 hours. Re-oxidation wasverified using SDS-PAGE under non-reducing conditions. Excess DHA wasremoved via 30 Kd MWCO cutoff centrifugal filter (Amicon Ultra-15, MerckMillipore, Cat No: UFC903024).

Conjugation to CDN-A: (Type I for reduced antibody) The reduced antibodyreaction mixture was cooled at 4° C. for 20 min. CDN-A-Linker (4.8 molarequivalents to antibody) in histidine buffer (20 mM, pH 7.4) was addedand incubated at 4° C. for 80 min. (Type II for re-oxidized antibody)The re-oxidized antibody was mixed with 10 molar equivalents ofCDN-A-linker and incubated at room temperature for 4 hours. Afterconjugation, the reaction was quenched with N-acetyl cysteine, and theexcess CDN-A-linker and N-acetyl cysteine were removed with a 5 mldesalting column (GE) under control of a FPLC system in PBS. The ADCsproduced were concentrated and stored at 4° C.

DAR Measurement

To determine DAR of ADCs carrying CDN-A as CDN, OD260/OD280 ratios weremeasured on a UV spectrometer and compared to a standard curve generatedusing mixtures of CDN-A and antibody at known ratios. As shown in thefollowing table, the DAR value of exemplary ADCs ranged from 1 to 6.3.The DAR value of ADCs using the Ab-A2 antibody (mu-anti-PDL1-cys) havingan additional cysteine by substitution showed a greater DAR value thanusing the wild-type Ab-A1 antibody (mu-anti-PDL1).

TABLE 1 Exemplary CDN-A ADCs ADC ID Ab ID Ab Notes CDN DAR ADC-IV Ab-B1mu-anti-EGFR (BioXcell BE0279) CDN-A 1 ADC-V Ab-A3 rt-anti-PDL1(BioXcell BE0101) CDN-A 1 ADC-VI Ab-A2 mu-anti-PDL1-cys CDN-A 2~4ADC-VII Ab-B2 mu-anti-EGFR-cys CDN-A 2 ADC-VIII Ab-B3 hu-anti-EGFR CDN-A6.3 ADC-IX Ab-B4 hu-anti-EGFR-A/C, V/C CDN-A 3 ADC-X Ab-C1 hu-anti-HER2(trastuzumab) CDN-A 4.8 ADC-XI Ab-D1 rt-anti-CD47 (BioXCell BE0270)CDN-A 2.5

Example 5. Preparation of CDN-B ADCs

Synthesis of 2,5-dioxopyrrolidin-1-yl4-(pyridin-2-yldisulfaneyl)pentanoate (linker I)

To a solution of 4-(pyridin-2-yldisulfaneyl) pentanoic acid (24 mg, 0.1mmol) and NHS (14 mg, 0.12 mmol) in DMA is added EDC (HCl salt, 61 mg,0.32 mmol). The solution is stirred at room temperature overnight. Afterfiltration, the filtrate is concentrated and purified by column(MeOH/DCM=0% to 10%) to obtain 2,5-dioxopyrrolidin-1-yl4-(pyridin-2-yldisulfaneyl)pentanoate as white solid (8 mg, 23.5%). (MS:[M+1]⁺ 341.1).

Conjugation of Antibodies to CDN-B

0.2 ml of 3 mM solution of2-amino-9-[(14S,15R,16R,17R,18S,19R,20R,21R)-20-(6-aminopurin-9-yl)-17,44,45-trihydroxy-44,45-dioxo-16-(2-sulfanylethyl)-38,39,40,41,42,43-hexaoxa-44,45diphosphatricyclooctadecan-2l-yl]-1H-purin-6-onein phosphate buffer pH 6.0 was mixed with 0.2 ml of 2 mM2,5-dioxopyrrolidin-1-yl 4-(pyridin-2-yldisulfaneyl)pentanoate in DMA.After incubation at room temperature overnight, 3.6 mg of target-bindingantibody in 2 ml PBS was added and incubated at room temperature foranother 2 hours. The mixture was concentrated and the conjugate waspurified on a 5 ml desalting column controlled on a FPLC systemequilibrated with PBS. The ADCs produced were concentrated and stored at4° C.

DAR Measurement

To release CDN-B from ADCs, DTT was added to ADC to a finalconcentration of 10 mM, incubated at 95° C. for 5 min, and passedthrough a 30 KD cut-off filter. The filtrate was diluted and added toTHP1-Lucia ISG cells permeabilized with 0.5 nM of Perfringolysin (PFO).16 hours later, luciferase activity was measured, concentration ofactive compounds released from ADC and DAR values was calculated bycomparing to standard curves. As shown in the following table, the DARvalue of exemplary ADCs ranged from 0.33 to 1.66.

TABLE 2 Exemplary CDN-B ADCs ADC ID Ab ID Ab Notes CDN DAR ADC-I Ab-A1mu-anti-PDL1 CDN-B 1.66 ADC-II Ab-A3 rt-anti-PDL1 (BioXcell BE0101)CDN-B 0.36 ADC-III Ab-B1 mu-anti-EGFR (BioXcell BE0279) CDN-B 0.33

Example 6. Cellular Activity of CDN-A and CDN-B ADCs

IFN Stimulatory Activity

Two reporter cell lines, mouse macrophage RAW-Lucia ISG and humanmonocyte THP1-Lucia ISG were used to assess activity of ADCs. Both celllines harbor IFN-stimulated response elements (ISRE) fused to an ISG54minimal promoter. ADCs at different concentrations (0.3, 1.0, and/or 3μM) were added to reporter cells and incubated for 16 h. Luciferaseactivity representing induction of interferon expression was compared toserial dilutions of a standard compound (cGAMP).

ADC-I through ADC-XI demonstrated effective interferon stimulatoryactivity of varying potencies, as summarized in Table 3. The reported“cGAMP equivalent” value is defined as the concentration of cGAMP (M)that is required to induce the same level of response induced by 1 μM ofADC.

TABLE 3 Potency of IFN Stimulatory Activity in Reporter Cells mousehuman RAW-Lucia ISG THP1-Lucia ISG ADC ID (cGAMP equivalent) (cGAMPequivalent) ADC-I 89 28 ADC-II 3.4 4.2 ADC-III ~7 0.58 ADC-IV ~10 weakADC-V ~10 weak ADC-VI >100 weak ADC-VII 3.1 n.d. ADC-VIII 33 n.d. ADC-IX31 n.d. ADC-X 10 n.d. ADC-XI 3 n.d.

All of the ADCs demonstrated potent interferon stimulatory activity inthe mouse RAW-Lucia ISG reporter assay (Table 3). ADC-I and ADC-VI wereof the highest potency (FIGS. 1B and 6B, respectively), followed byADC-VIII, ADC-IX (FIGS. 8B and 9B, respectively), then by ADC-IV, ADC-V,and ADC-X (FIGS. 4B, 5B, and 10, respectively), and then by ADC-III(FIG. 3B), ADC-II (FIG. IIB), and ADC-XI. As shown in FIGS. 3B, 4B, 5B,and 6B, treatment with the disclosed ADCs demonstrate synergism ofinterferon stimulatory activity when compared to treatment with theantibody alone, or the CDN alone, or a combined value of both theantibody and agonist potencies.

CDN-A antibody conjugates also demonstrated interferon stimulatoryactivity in a reporter assay using human monocyte THP1-Lucia ISG cells(Table 3). Of those tested, ADC-I was of the highest potency (FIG. 1C),followed by ADC-II (FIG. 1C), and ADC-III.

The activities of ADC-VIII and ADC-IX were further tested in aTHP1-lucia ISG cell line stably expressing human EGFR. Both ADCsexhibited strong interferon-stimulatory activities in these cells (Table4). The reported “cGAMP equivalent” value is defined as theconcentration of cGAMP (nM) that is required to induce the same level ofresponse induced by 1 nM of ADC. EC50 is half maximal effectiveconcentration.

TABLE 4 Activity of ADC-VIII and ADC-IX in THP1-EGFR-lucia ISG cells ADCID cGAMP equivalent EC50 ADC-VIII 977 11.7 nM ADC-IX 912  8.2 nM

Example 7. Antitumor Efficacy of CDN-A and CDN-B ADCs

Antitumor efficacy of selected ADCs was tested in mouse syngeneic tumormodels B16-F10 metastatic melanoma, human EGFR-transfected B16F10(B16F10-EGFR), and human HER2-transfected Lewis lung carcinoma(LLC1-HER2). Briefly, 10⁶ of log-phase tumor cells in 100 μL of PBS wereinjected subcutaneously into C57BL6 mice at their right flanks. Four tosix days later, when tumor volumes were 50-100 mm³, mice were regroupedaccording to their tumor sizes and treated intraperitoneally with 50 to200 μg of ADC, or with unconjugated antibody, unconjugated CDN, or mockPBS as controls (see figure legends for treatment details).

Tumor volumes and mice survival were monitored. ADC-IV, ADC-VI, ADC-VII,ADC-VIII, and ADC-IX demonstrated strong anti-tumor efficacy in bothregression and survival and are potential candidates to treat humantumors. ADC-X was shown to slow tumor progression. ADC-I was also shownto slow tumor progression, but toxicity was observed.

Tumor Volume

In mice bearing B16F10 tumors, treatment with ADC-I (anti-PDL1-CDN-B)slowed tumor progression compared to mock, anti-PDL1 antibody alone,CDN-B alone, and the combination of anti-PDL1 antibody and unconjugatedCDN-B (FIG. 1C).

In mice bearing B16F10-EGFR tumors, treatment with ADC-IV(anti-EGFR-CDN-A) slowed tumor progression and reduced tumor volumecompared to mock treatment or treatment with anti-PDL1 antibody (FIGS.4C and 4E). Comparative treatment with anti-EGFR antibody plusunconjugated CDN failed to stop tumor growth (FIG. 4C), suggestingimproved efficacy when using an ADC for targeted CDN delivery. WhenADC-IV was combined with anti-PDL1 antibody a complete suppression oftumor expansion was observed (FIG. 4E), suggesting improved efficacywhen combining a CDN ADC with checkpoint inhibitors.

In mice bearing B16F10-EGFR tumors, ADC-VI (anti-PDL1-CDN-A)administered intraperitoneally at a dose of 200 μg (FIG. 6C) orintratumorally at a dose of 10 g or 50 μg (FIG. 6D) prevented tumorexpansion. Within two weeks after treatment with ADC-VI, B16F10-EGFRtumors showed complete remission, whereas treatment with the anti-PDL1antibody alone or mock failed to stop tumor expansion.

In mice bearing B16F10-EGFR tumors, ADC-VII (anti-EGFR-CDN-A) led to amarked reduction of tumor expansion compared to mock treatment ortreatment with anti-PDL1 antibody (FIGS. 7C and 7E). Comparativetreatment with anti-EGFR antibody plus unconjugated CDN failed to stoptumor growth (FIG. 7C), suggesting improved efficacy when using an ADCfor targeted CDN delivery. Enhancement of tumor suppression was observedwhen ADC-VII was combined with anti-PDL1 antibody, whereas treatmentwith anti-PDL1 antibody alone or mock failed to stop tumor expansion(FIG. 7E), suggesting improved efficacy when combining a CDN ADC withcheckpoint inhibitors.

In mice bearing B16F10-EGFR tumors, ADC-VIII (anti-EGFR-CDN-A) led to amarked reduction of tumor expansion (FIG. 8C). This effect was enhancedwhen ADC-VIII was combined with anti-PDL1 antibody, whereas treatmentwith anti-PDL1 antibody alone or mock failed to stop tumor expansion,suggesting improved efficacy when combining a CDN ADC with checkpointinhibitors.

In mice bearing B16F10-EGFR tumors, ADC-IX (anti-EGFR-CDN-A) led to areduction of tumor expansion, and a similar effect was observed whenADC-IX was combined with anti-PD-L1 antibody (FIG. 9C). Treatment withanti-PDL1 antibody alone or mock failed to stop tumor expansion.

In mice bearing LLC1-HER2 tumors, ADC-X (anti-HER2-CDN-A) reduced tumorexpansion in two separate mouse studies (FIGS. 10C and 10E), whereastreatment with the anti-PDL1 antibody alone or mock failed to stop tumorexpansion. Comparative treatment with anti-HER2 antibody plusunconjugated CDN failed to stop tumor growth, suggesting improvedefficacy when using an ADC for targeted CDN delivery. FIG. 10E furthershows that intratumoral administration of ADC-X at a dose of 30 μgresulted in complete tumor remission.

Survival

The survival of mice bearing B16F10-EGFR or LLC1-HER2 tumors wasmonitored. Treatment with ADC-IV (anti-EGFR-CDN-A) increased the time ofsurvival of B16F10-EGFR tumor bearing mice (3/5 at Day 37, FIG. 4D; 0/5at Day 39, FIG. 4F) compared to treatment with anti-PDL1 antibody alone(0/5 at Day 26, FIG. 4D; 0/5 at Day 29, FIG. 4F), and mock (0/5 at Day23, FIG. 4D; 0/5 at Day 20, FIG. 4F). Survival following ADC-IVtreatment exceeded that of comparative treatment using anti-EGFRantibody plus unconjugated CDN (0/5 at Day 34, FIG. 4D), suggestingimproved efficacy when using an ADC for targeted CDN delivery. Acombination treatment of ADC-IV with anti-PDL1 antibody improvedsurvival to 80% of mice (4/5 at Day 42), suggesting improved efficacywhen combining a CDN ADC with checkpoint inhibitors.

Treatment with ADC-VI (anti-PDL1-CDN-A) administered intraperitoneallyled to total survival of B16F10-EGFR tumor bearing mice (5/5 at Day 43),compared to treatment with the anti-PDL1 antibody alone (0/5 at Day 29),or mock (0/5 at Day 27) (FIG. 6D). Likewise, treatment of ADC-VIadministered intratumorally led to total survival of the tumor bearingmice (FIG. 6F).

Treatment with ADC-VII (anti-EGFR-CDN-A) increased survival ofB16F10-EGFR tumor bearing mice at endpoint (2/5 at Day 36, FIGS. 7D and7F) compared to treatment with anti-PDL1 antibody alone (0/5 at Day 27,FIG. 7F), or mock (0/5 at Day 23, FIGS. 7D and 7F). Survival followingADC-VII treatment exceeded that of comparative treatment using anti-EGFRantibody plus unconjugated CDN (0/5 at Day 22, FIG. 7D), suggestingimproved efficacy when using an ADC for targeted CDN delivery. Acombination treatment of ADC-VII with anti-PDL1 antibody improvedsurvival to 60% of mice (3/5 at Day 36, FIG. 7F) compared to ADC-VII,suggesting improved efficacy when combining a CDN ADC with checkpointinhibitors.

Treatment with ADC-VIII (anti-EGFR-CDN-A) increased survival ofB16F10-EGFR tumor bearing mice (1/5 at Day 35) compared to treatmentwith anti-PDL1 antibody alone (0/5 at Day 27), or mock (0/5 at Day 25)(FIG. 8D). Treatment with a combination of ADC-VIII and anti-PD-L1antibody improved survival to 80% of mice (4/5 at Day 35), suggestingimproved efficacy when combining a CDN ADC with checkpoint inhibitors.

Treatment with ADC-IX (anti-EGFR-CDN-A) increased survival ofB16F10-EGFR tumor bearing mice (3/5 at Day 30) compared to treatmentwith anti-PDL1 antibody alone (0/5 at Day 23), or mock (0/5 at Day 19)(FIG. 9D). Treatment with a combination of ADC-IX and anti-PD-L1antibody improved survival to 60% of mice (3/5 at Day 30), suggestingimproved efficacy when combining a CDN ADC with checkpoint inhibitors.

Treatment with ADC-X (anti-HER2-CDN-A) increased the time of survival ofLLC1-HER2 tumor bearing mice (0/5 at Day 41) compared to treatment withmock (0/5 at Day 32) (FIG. 10D). Survival time following ADC-X treatmentexceeded that of comparative treatment using anti-HER2 antibody plusunconjugated CDN (0/5 at Day 32), suggesting improved efficacy whenusing an ADC for targeted CDN delivery.

The invention claimed is:
 1. A compound of the formula L-CDN, wherein Lis a linker that includes a site capable of coupling to a complementarysite on an antibody or antigen-binding fragment; CDN is a cyclicdinucleotide having the structure of Formula IIk:

wherein W, X, Y, and Z are independently CH or N; R¹ is C₂₋₄alkylsubstituted with a thiol, amino, an amino or C₁₋₆alkylamino group; R^(P)is, independently for each occurrence, hydroxyl, thiol, C₁₋₆alkyl, —BH₃⁻, or —NR′R″, wherein R′ and R″ are, independently for each occurrence,hydrogen or C₁₋₆alkyl optionally substituted with one or more groupsselected from halogen, thiol, hydroxyl, carboxyl, C₁₋₆alkoxy,C₁₋₆hydroxyalkoxy, —OC(O)C₁₋₆alkyl, —N(H)C(O)C₁₋₆alkyl,—N(C₁₋₃alkyl)C(O)C₁₋₆alkyl, amino, C₁₋₆alkylamino, di(C₁₋₆alkyl)amino,oxo, and azido; or R′ and R″ on the same nitrogen together form aC₃₋₅heterocyclic ring; or a pharmaceutically acceptable salt thereof;and CDN is coupled to L at the amino or C₁₋₆alkylamino group of R¹. 2.The compound of claim 1, wherein CDN is coupled to L via an amide, acarbamate, or a urea group.
 3. The compound of claim 2, wherein thecompound has the structure of Formula IXb:

wherein R^(L) represents the remainder of the linker L.
 4. The compoundof claim 1, wherein R^(P), independently for each occurrence, ishydroxyl or thiol.
 5. The compound of claim 1, wherein Y and W are bothCH.
 6. The compound of claim 1, wherein X and Z are both N.
 7. Thecompound of claim 1, wherein R¹ is ethyl substituted with an aminogroup.
 8. The compound of claim 1, wherein R¹ is ethyl substituted witha C₁₋₆alkylamino group.
 9. The compound of claim 8, wherein R¹ is ethylsubstituted with a methylamino group.
 10. The compound of claim 1,wherein at least one occurrence of R^(P) is hydroxyl.
 11. The compoundof claim 10, wherein both occurrences of R^(P) are hydroxyl.
 12. Thecompound of claim 10, wherein one occurrence of R^(P) is hydroxyl andthe other is thiol.
 13. The compound of claim 1, wherein at least oneoccurrence of R^(P) is thiol.
 14. The compound of claim 13, wherein bothoccurrences of R^(P) are thiol.
 15. The compound of claim 1, wherein theCDN has the following structure:

or a pharmaceutically acceptable salt thereof.
 16. The compound of claim2, wherein CDN is coupled to L via a carbamate group.
 17. The compoundof claim 16, wherein the compound has the following structure:


18. The compound of claim 16, wherein the CDN has the followingstructure:

or a pharmaceutically acceptable salt thereof.
 19. The compound of claim2, wherein the compound has the structure of Formula IXa:

wherein R^(L) represents the remainder of the linker L.
 20. The compoundof claim 2, wherein CDN is coupled to L via an amide group.
 21. Thecompound of claim 20, wherein the CDN has the following structure:

or a pharmaceutically acceptable salt thereof.
 22. The compound of claim2, wherein the compound has the structure of Formula IXc:

wherein R^(L) represents the remainder of the linker L.
 23. The compoundof claim 2, wherein CDN is coupled to L via a urea group.
 24. Thecompound of claim 23, wherein the CDN has the following structure:

or a pharmaceutically acceptable salt thereof.
 25. A compound of theformula L-CDN, wherein L is a linker that includes a site capable ofcoupling to a complementary site on an antibody or antigen-bindingfragment; CDN is a cyclic dinucleotide having the structure of FormulaIIk:

wherein W, X, Y, and Z are independently CH or N; R¹ is C₂₋₄alkylsubstituted with an amino group; R^(P) is, independently for eachoccurrence, hydroxyl or thiol; or a pharmaceutically acceptable saltthereof; and CDN is coupled to L at the amino group of R¹ via an amide,a carbamate, or a urea group.
 26. The compound of claim 25, wherein R¹is ethyl substituted with an amino group.